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Due to the promising applications of propolis as a natural food preservative, functional ingredient or supplement, on one side, and the inherent toxicity and high volatility of organic solvents used for extraction of phenolics from propolis, a range of natural eutectic solvents (NESs) was evaluated for the extraction of phenolic compounds from poplar type propolis to create “green-labelled” food products. The phenolic profile of novel green propolis extracts was evaluated using high-performance thin-layer chromatography, while 16 phenolic compounds were quantified by ultra-high-performance liquid chromatography hyphenated with mass spectrometry. Pinocembrin and chrysin were the most dominant phenolics with an average concentration of 3111 and 3039 mg/kg in lactic acid-based NES, respectively. Biological activity, such as antioxidative and antimicrobial activities, were determined using spectrophotometric and well diffusion methods. Considering all spectrophotometric assays lactic acid-based presented as the best extraction medium for phenolic extraction, with TPC, TFC and RSA values 150.0 mg GAE/g, 190.0 mg RUE/g, and 132.8 mg TE/g on average, respectively. From 38 studied NESs, choline-chloride:tartaric acid (1:1 n/n), glycerol:lactic acid (1:1 n/n) and glycine: lactic acid (1:3 n/n) were the most promising extraction medium for obtaining the propolis extracts enriched with bioactive compounds. Additionally, proposed green solvents were proved to be the designer solvents and showed potential to be tuned to target specific phenolic compounds. Improved, or at least maintained, biological activity of NES propolis extracts support its application in agriculture industry.

Due to the promising applications of propolis as a natural food preservative, functional ingredient or supplement, on one side, and the inherent toxicity and high volatility of organic solvents used for extraction of phenolics from propolis, a range of natural eutectic solvents (NESs) was evaluated for the extraction of phenolic compounds from poplar type propolis to create “green-labelled” food products. The phenolic profile of novel green propolis extracts was evaluated using high-performance thin-layer chromatography, while 16 phenolic compounds were quantified by ultra-high-performance liquid chromatography hyphenated with mass spectrometry. Pinocembrin and chrysin were the most dominant phenolics with an average concentration of 3111 and 3039 mg/kg in lactic acid-based NES, respectively. Biological activity, such as antioxidative and antimicrobial activities, were determined using spectrophotometric and well diffusion methods. Considering all spectrophotometric assays lactic acid-based presented as the best extraction medium for phenolic extraction, with TPC, TFC and RSA values 150.0 mg GAE/g, 190.0 mg RUE/g, and 132.8 mg TE/g on average, respectively. From 38 studied NESs, choline-chloride:tartaric acid (1:1 n/n), glycerol:lactic acid (1:1 n/n) and glycine: lactic acid (1:3 n/n) were the most promising extraction medium for obtaining the propolis extracts enriched with bioactive compounds. Additionally, proposed green solvents were proved to be the designer solvents and showed potential to be tuned to target specific phenolic compounds. Improved, or at least maintained, biological activity of NES propolis extracts support its application in agriculture industry.

Cover crops play an important role in low-input cropping systems, increasing the use of agro-ecosystem services. Due to the lack of information about the impact of cover crops and bio-fertilizers on popcorn maize (Zea mays everta Sturt.) growth and yield quality, especially the popping volume and nutritive quality, such as concentrations of protein and mineral elements, this research aimed to provide essential information. The interrelation between popcorn maize productivity and quality with important groups of soil microorganisms presents additional novelty. The results demonstrated that field pea is a beneficial cover crop, especially when combined with a bio-fertilizer, supporting the accumulation of maize biomass, chlorophyll, yield potential, and the concentrations of protein, Ca, Mg, Fe, and Zn. In addition, field pea residues promoted N-fixing bacteria, and the number of total microorganisms, especially actinomycetes and decomposing bacteria, which could promote nutrient uptake and grain quality. Residues of cover crop mixtures, common vetch + winter oats and field pea + winter oats, promoted the total number of microorganisms in the soil, and up to the end of vegetation, a greater number of decomposition and ammonification microorganisms were found, especially when the bio-fertilizer was applied, which consequently could support greater maize biomass. Popping volume, as a main trait of popcorn maize, had the highest value in the common vetch + winter oats variant, supporting again the statement that quality traits could be enhanced in sustainable production. Unlike living cover crops, mulch mainly affected soil microbial communities and promoted the development of actinomycetes and cellulolytic microorganisms during the growing season. The results of this research could contribute to the development of sustainable popcorn maize production for improved grain quality. They could also serve as a basis for isolating beneficial soil microorganisms to develop new

Current knowledge of the microbiota of soda pans of the Carpathian Basin focuses mainly on diatoms. In studying six soda pans in Vojvodina (Serbia), we aimed to gain deeper insights into the diatom community by using not only traditional microscopic analysis but also high-throughput sequencing of the 18S rRNA gene. In addition, this study aimed to evaluate bacterial communities in these soda pans for the first time using the 16S rRNA gene marker. Although similar numbers of diatom taxa were found using both approaches, a significant discrepancy in species composition was found. The genus Nitzschia was one of the most represented and numerous genera according to the morphological and molecular analyses, but with lower diversity according to the molecular data, with the sequences mostly unassigned. Another major discrepancy was noticed with taxon Halamphora veneta, the results of metabarcoding showed high abundance in almost all soda pans, while microscopy detected it as very low. Diverse communities of aerobic and anaerobic bacteria were detected in the studied soda pans, such as Proteobacteria, Bacteroidota, Actinobacteria, Bacillaceae, and Peptoclostridium. According to current knowledge, harsh environmental conditions and habitat homogeneity regulate diatom and bacteria community composition and enable the survival of taxa with predominantly high ecophysiological plasticity. Finally, uncovering the structure of the microbial community as the first phase in developing an efficient tool to assess the ecological status of these fragile ecosystems is an essential step towards their conservation.

Cover crops play an important role in low-input cropping systems, increasing the use of agro-ecosystem services. Due to the lack of information about the impact of cover crops and bio-fertilizers on popcorn maize (Zea mays everta Sturt.) growth and yield quality, especially the popping volume and nutritive quality, such as concentrations of protein and mineral elements, this research aimed to provide essential information. The interrelation between popcorn maize productivity and quality with important groups of soil microorganisms presents additional novelty. The results demonstrated that field pea is a beneficial cover crop, especially when combined with a bio-fertilizer, supporting the accumulation of maize biomass, chlorophyll, yield potential, and the concentrations of protein, Ca, Mg, Fe, and Zn. In addition, field pea residues promoted N-fixing bacteria, and the number of total microorganisms, especially actinomycetes and decomposing bacteria, which could promote nutrient uptake and grain quality. Residues of cover crop mixtures, common vetch + winter oats and field pea + winter oats, promoted the total number of microorganisms in the soil, and up to the end of vegetation, a greater number of decomposition and ammonification microorganisms were found, especially when the bio-fertilizer was applied, which consequently could support greater maize biomass. Popping volume, as a main trait of popcorn maize, had the highest value in the common vetch + winter oats variant, supporting again the statement that quality traits could be enhanced in sustainable production. Unlike living cover crops, mulch mainly affected soil microbial communities and promoted the development of actinomycetes and cellulolytic microorganisms during the growing season. The results of this research could contribute to the development of sustainable popcorn maize production for improved grain quality. They could also serve as a basis for isolating beneficial soil microorganisms to develop new

The pygidial glands of carabids produce strong-smelling vapours. In this study, we examined the chemical composition of the gland secretions and the structure of the glands in five species of Carabini ground beetles (one species from the subtribe Calosomatina and four species from the subtribe Carabina): Calosoma (Calosoma) maderae (Fabricius, 1775), Carabus (Carabus) granulatus Linnaeus, 1758, C. (Limnocarabus) clathratus Linnaeus, 1760, C. (Carabus) ulrichii Germar, 1823, and C. (Procerus) gigas Creutzer, 1799. Additionally, we tested the antibacterial potential of the pygidial gland secretions of the two latter species against 11 bacterial strains. In order to detect the chemical content of the secretions, we used gas chromatography–mass spectrometry (GC–MS). The secretion extracts were applied against selected strains of medically important bacteria. We used bright-field microscopy to examine the morphology of the glands. We discovered a total of 11 chemical compounds in the pygidial gland extracts of the ground beetles we analysed. Ten of these compounds were identified as seven carboxylic acids, two hydrocarbons, and one aromatic aldehyde, while one chemical remained unidentified. Most of the components were isolated from the secretion of C. (L.) clathratus (nine), while the lowest number of compounds was found in C. (P.) gigas (two). Methacrylic acid was the most dominant compound by percentage in all five species, while angelic acid was also detected in all samples. As expected, salicylaldehyde was exclusively found in the species of the genus Calosoma Weber, 1801. The secretion of C. (P.) gigas was shown to achieve the highest level of antibacterial activity against Pseudomonas aeruginosa, Salmonella enterica, and S. typhimurium (even the same level as the positive control streptomycin), while the secretion of C. (C.) ulrichii achieved the highest antimicrobial potential against Staphylococcus epidermidis, S. aureus, Listeria monocytogenes, and Bacillus cer

In the present study, we investigated in vitro the antimicrobial activity of the pygidial gland secretion of the guanophilic ground beetle Laemostenus (Pristonychus) punctatus (Dejean, 1828) and some of its chemicals against resistant and non-resistant bacteria and Candida species, the synergistic and additive potential of combinations of selected chemicals and antimicrobial drugs against resistant bacterial and fungal strains, anti-adhesive and anti-invasive potential of the secretion and formic acid alone and in selected combinations with antimicrobial drugs against methicillin-resistant Staphylococcus aureus (MRSA) toward spontaneously immortalized human keratinocyte cell line (HaCaT cells). In addition, we examined the antiproliferative activity of the secretion and formic acid in vitro. The tested secretion and the standards of formic and oleic acids possessed a significant level of antimicrobial potential against all tested strains (P < 0.05). The isolate from guano Pseudomonas monteilii showed the highest resistance to the secretion and formic acid, while MRSA achieved a significantly high level of susceptibility to all agents tested, particularly to the combinations of formic acid and antibiotics, but at the same time showed a certain level of resistance to the antibiotics tested individually. Candida albicans and C. tropicalis were found to be the most sensitive fungal strains to the secretion. Formic acid (MIC 0.0005 mg/mL) and gentamicin (MIC 0.0010 mg/mL) in the mixture achieved synergistic antibacterial activity against MRSA (FICI = 0.5, P < 0.05). The combination of formic acid, gentamicin and ampicillin accomplished an additive effect against this resistant bacterial strain (FICI = 1.5, P < 0.05). The secretion achieved a better inhibitory effect on the adhesion ability of MRSA toward HaCaT cells compared to formic acid alone, while formic acid showed better results regarding the invasion (P < 0.001). The combinations of gentamicin and ampicillin, as

Natural Deep Eutectic Solvents (NADES) are novel and promising solvents for green extraction of phytochemicals from food and agricultural products. NADES are made of natural origin compounds that are connected via hydrogen bond and have unique tuneable properties1. Numerous studies investigated the extraction of bioactive compounds using NADES resulted in better extraction efficiency compared to conventional solvents, suggesting large potential of these solvents2,3. Low-toxicity, natural origin and affordability of NADES-based extracts enables their application as a preservative in the food industry or functional ingredient of food supplements. The aim of this study was to develop an efficient eco-friendly method for the extraction of phenolic compounds from berries, blueberry, chokeberry and black goji berry, using NADES. This goal will be achieved through investigation of extraction efficiency of applied NADES based on three different approaches, a) chromatographic, through high-performance thin layer chromatography (HPTLC) fingerprint analysis, ultra-high-performance liquid chromatography with a diode array detector and a triple-quadrupole mass spectrometer (UHPLC-DAD-MS/MS) target analysis and bioautography, b) spectroscopic, through quality control parameters, total phenolic content (TPC), total flavonoid content (TFC) and radical scavenging activity (RSA), and c) microbiological, through well diffusion method and minimum inhibitory concentration (MIC). In this study, 36 NADES mixtures, prepared from primary plant metabolites, were tested as green alternatives for extraction of phenolics from three berries. Different hydrogen-bond acceptors (HBA), such as choline chloride, L-proline, L-glycine, and L-lysine were mixed with various hydrogen-bond donors (HBD), four organic acids, two sugars, glycerol and urea, at various molar ratios for NADES preparation. Guided by the fact that extraction efficiency depends on polarity, viscosity and dissolving ability of solve

Introduction: A novel group of Rhizobiumspp. strains belonging to the “tumorigenes” clade has recently been described on blackberry in Serbia and Germany and on rhododendron in Germany. The BIOCTA project aimed to characterize efficient plant-associated bacterial strains for biocontrol of crown gall, thus providing an environmentally friendly alternative to pesticides that would contribute to the development of sustainable agriculture. Methods: Antagonistic potential of 37 biocontrol strains against two R. tumorigenes strains 932 and 1078 and Rhizobium sp. strain rho-6.2 was evaluated in vitro using the “well diffusion” method, as well as in vivo on tomato plants, using two inoculation strategies (co-inoculation and preventive). DNA metabarcoding approach was used to analyze the phytobiome of treated and non-treated tomato plants. Results: Based on the determined in vitro antagonistic potential, seven strains – Bacillus spp. (B. amyloliquefaciens ID084 and GT28.3, B. velezensis X5-2, and B. subtilis GD1), Pseudomonas sp. (R-6.10 and R11-20) and Agrobacterium rosae rho-6.1 were selected for further in vivo experiments. Of all tested strains/treatments, two Pseudomonas strains were the most efficient, showing up to 92.86%efficacy in suppressing tumors caused by Rhizobiumsp. strain rho-6.2 when applied in a co-inoculation strategy. Based on the DNA metabarcoding analysis, genera Pseudolabrys and Asanoa prevailed in the co-inoculation strategy, while Allorhizobium-Neorhizobium-Pararhizobium-Rhizobium prevailed in positive control. Conclusion: Crown gall tumors have shown to be a valuable source of antagonistic isolates. Pseudomonas strains R-6.10 and R11-20 could be proposed for the efferent control of crown gall caused by newly described Rhizobium spp. strains in nurseries.

Targeted physiological groups of bacteria were cultivated and identified in the brackish groundwaters of Obrenovačka Banja (OB), Lomnički Kiseljak (LK) and Velika Vrbnica (VV) using biological activity reaction tests (BARTs) to assess the biocorrosion, biofouling and health risks. The highest density of iron-related, sulfate-reducing, slime-forming, facultatively anaerobic heterotrophic, denitrifying bacteria and representatives of Pseudomonas spp. was recorded in the OB sample, while the lowest density of the same physiological groups of bacteria was recorded in the LK sample. Facultatively anaerobic heterotrophic bacteria were the most abundant in the OB and LK samples, while, in contrast, heterotrophic aerobic bacteria were the most abundant in the VV sample. All tested samples were characterized by a high degree of biochemical activity associated with iron-related, sulfate-reducing, slime-forming, heterotrophic aerobic and facultatively anaerobic bacteria. Also, high biochemical activity of denitrifying bacteria was recorded in the OB sample, and the same activity of Pseudomonas species was recorded in the OB and VV samples. For OB and LK groundwaters, the highest degree of risk was estimated for biocorrosion process, while for the OB and VV occurrences, the highest degree of risk was estimated for biofouling process. The health risk was present for all examined groundwaters. Caution is warranted in further use of all investigated occurrences due to the established public health risk and an immediate revitalization of the OB, LK and VV wells is necessary.

The pygidial glands of carabids produce strong-smelling vapours. In this study, we examined the chemical composition of the gland secretions and the structure of the glands in five species of Carabini ground beetles (one species from the subtribe Calosomatina and four species from the subtribe Carabina): Calosoma (Calosoma) maderae (Fabricius, 1775), Carabus (Carabus) granulatus Linnaeus, 1758, C. (Limnocarabus) clathratus Linnaeus, 1760, C. (Carabus) ulrichii Germar, 1823, and C. (Procerus) gigas Creutzer, 1799. Additionally, we tested the antibacterial potential of the pygidial gland secretions of the two latter species against 11 bacterial strains. In order to detect the chemical content of the secretions, we used gas chromatography–mass spectrometry (GC–MS). The secretion extracts were applied against selected strains of medically important bacteria. We used bright-field microscopy to examine the morphology of the glands. We discovered a total of 11 chemical compounds in the pygidial gland extracts of the ground beetles we analysed. Ten of these compounds were identified as seven carboxylic acids, two hydrocarbons, and one aromatic aldehyde, while one chemical remained unidentified. Most of the components were isolated from the secretion of C. (L.) clathratus (nine), while the lowest number of compounds was found in C. (P.) gigas (two). Methacrylic acid was the most dominant compound by percentage in all five species, while angelic acid was also detected in all samples. As expected, salicylaldehyde was exclusively found in the species of the genus Calosoma Weber, 1801. The secretion of C. (P.) gigas was shown to achieve the highest level of antibacterial activity against Pseudomonas aeruginosa, Salmonella enterica, and S. typhimurium (even the same level as the positive control streptomycin), while the secretion of C. (C.) ulrichii achieved the highest antimicrobial potential against Staphylococcus epidermidis, S. aureus, Listeria monocytogenes, and Bacillus cer

In the present study, we investigated in vitro the antimicrobial activity of the pygidial gland secretion of the guanophilic ground beetle Laemostenus (Pristonychus) punctatus (Dejean, 1828) and some of its chemicals against resistant and non-resistant bacteria and Candida species, the synergistic and additive potential of combinations of selected chemicals and antimicrobial drugs against resistant bacterial and fungal strains, anti-adhesive and anti-invasive potential of the secretion and formic acid alone and in selected combinations with antimicrobial drugs against methicillin-resistant Staphylococcus aureus (MRSA) toward spontaneously immortalized human keratinocyte cell line (HaCaT cells). In addition, we examined the antiproliferative activity of the secretion and formic acid in vitro. The tested secretion and the standards of formic and oleic acids possessed a significant level of antimicrobial potential against all tested strains (P < 0.05). The isolate from guano Pseudomonas monteilii showed the highest resistance to the secretion and formic acid, while MRSA achieved a significantly high level of susceptibility to all agents tested, particularly to the combinations of formic acid and antibiotics, but at the same time showed a certain level of resistance to the antibiotics tested individually. Candida albicans and C. tropicalis were found to be the most sensitive fungal strains to the secretion. Formic acid (MIC 0.0005 mg/mL) and gentamicin (MIC 0.0010 mg/mL) in the mixture achieved synergistic antibacterial activity against MRSA (FICI = 0.5, P < 0.05). The combination of formic acid, gentamicin and ampicillin accomplished an additive effect against this resistant bacterial strain (FICI = 1.5, P < 0.05). The secretion achieved a better inhibitory effect on the adhesion ability of MRSA toward HaCaT cells compared to formic acid alone, while formic acid showed better results regarding the invasion (P < 0.001). The combinations of gentamicin and ampicillin, as

Numerous microbial taxa are used as biofertilizers because they have the potential to competitively colonize the rhizosphere or root interior of plants and promote nutrient uptake. The aim was to investigate the shifts of keystone bacterial species in maize roots and rhizosphere during seedling stage, flowering, and harvest, under different treatments of bacterial phytobiotic (PHY), poultry manure (PM) and their combination (PHY_PM). Soil bacterial communities remained largely unchanged regardless of the treatment applied or phenophase studied, with uncultured Gaiellales and Bacillus being the most abundant. In contrast, bacterial communities in roots differed in terms of distribution and relative abundance (RA) of different taxa between growth stages and treatments. The most abundant bacterial taxa in the roots during the initial seedling stage was Pseudomonas. In the flowering, Bacillus occurred with a two- to threefold higher RA in treatments with PHY or PM compared to the negative control, while Lechevalieria dominated during the harvest. Differential abundance analysis at the seedling stage showed a reduction of Pseudomonas in roots treated with PHY, while Pseudonocardia was significantly more abundant in roots treated with PM than in the other samples. Massilia, Streptomyces, Lechevalieria, Microbacteriaceae, Aeromicrobium, Sphingomonas and Roseiflexaceae were significantly higher in the PHY_PM treated root samples. Steroidobacter and Bdellovibrio were absent in PHY and PM treated root samples during flowering, while Bdellovibrio was significantly reduced in PHY_PM treatment compared to the negative control. RA of Bradyrhizobium and Polaromonas was reduced in PHY treated root samples, and Dongia was completely absent. Gaiellales was significantly more abundant, while Sphingomonas was less abundant in roots treated with PM and PHY_PM. Solirubrobacter and Bdellovibrio were absent from roots during harvest in all three treatment types. Pajaroellobacter was comple

Soda pans are restricted to the Carpathian Basin and differ from other similar saline waters mainly due to the dominant presence of Na+, HCO3–, and CO32– ions. These shallow and temporary aquatic habitats are inhabited by a number of species that can thrive in extreme environments. In Serbia, saline habitats are rare, endangered, and most common in the territory of Vojvodina. One of the most investigated group of organisms in soda pans is diatoms. Due to ecophysiological plasticity, they can survive and tolerate extreme environmental conditions which gives them a competitive advantage over other algae. Quite the opposite, prokaryotes in saline habitats are not sufficiently studied and data are scarce. Thus, samples from 6 soda pans were collected in March 2021 aiming to determine diatom (microscopic and molecular analysis, 18S rRNA gene) and bacterial community (16S rRNA gene). Most of the recorded diatom taxa are neutrophilic to alkalibiontic, mostly halophilic, and eutrophic species. The species richness diversity index was the highest in Velika Slatina and Slatina, while it was lowest in Mala Rusanda. The genus Nitzschia was one of the most represented and numerous according to morphological analyses (24 taxa, 4 dominant), while molecular analyses indicate 11 different species with low abundances and mainly with unassigned sequences. The biggest discrepancy in these two approaches was observed in Mala Rusanda and Okanj bara. Metabarcoding analysis indicated Halamphora veneta as the dominant taxa, while morphological analyses have shown a predominance of Nitzschia supralitorea and Surirella brebissonii in Mala Rusanda, and N. austriaca in Okanj bara. Estimated richness and alpha diversity indices showed the highest bacterial diversity in Velika Rusanda and Okanj bara, while the lowest richness was in Mala Rusanda. Representatives of the phylum Proteobacteria were detected predominantly in Pečena Slatina, Bacteroidota in Velika Slatina, Actinobacteria in Okanj bara

This study aimed to investigate the prevalence and resistance mechanisms of colistin-resistant Acinetobacter baumannii (ColRAB) isolates in Serbia, assess their genetic relatedness to other circulating A. baumannii isolates in the neighbouring European countries, and analyse the global genomic epidemiology of ColRAB isolates. A total of 784 isolates of A. baumannii were recovered from hospitalised patients in Serbia between 2018 and 2021. The antimicrobial susceptibility testing was performed using disk diffusion and broth microdilution. All ColRAB isolates were subjected to DNA isolation and whole-genome sequencing (WGS). Overall, 3.94 % (n = 30) isolates were confirmed as ColRAB. Results of mutational and transcriptional analysis of genes associated with colistin resistance indicate the central role of the two-component regulating system, PmrAB, and increased expression of the pmrC gene in ColRAB. Most of the isolates (n = 29, 96.6 %) belonged to international clone II, with the most common sequence type being STPas2 (n = 23, 76.6 %). Based on the WGS analysis, ColRAB isolates belonging to the same ST isolated in various countries were grouped into the same clusters, indicating the global dissemination of several high-risk clonal lineages. Phylogenomic analysis of ColRAB isolates, together with all previously published A. baumannii genomes from South-Eastern European countries, showed that colistin resistance arose independently in several clonal lineages. Comparative genomic analysis revealed multiple genes with various roles (transcriptional regulation, transmembrane transport, outer membrane assembly, etc.), which might be associated with colistin resistance in A. baumannii. The obtained findings serve as the basis for further studies, contributing to a better understanding of colistin resistance mechanisms in A. baumannii.

“Pirot ‘ironed’ sausage“ (Pis) is a traditional, fermented sausage, made from different types of meat (beef and chevon), without additives or starter cultures. The physical–chemical properties (pH, water activity, fats, moisture, and protein contents) were examined in the initial meat batter stuffing and during ripening. Total bacterial diversity was examined at different time points using both culturable (traditional) and non-culturable (NGS sequencing) approaches. During the ripening, a decrease in pH value, aw, and moisture content was observed, as well as an increase in protein and fat content. At least a two-fold significant decrease was noted for colorimetric values during the ripening period. The dominance of Proteobacteria and Firmicutes was observed in the non-culturable approach in all studied samples. During the ripening process, an increase in Firmicutes (from 33.5% to 63.5%) with a decrease in Proteobacteria (from 65.4% to 22.3%) was observed. The bacterial genera that were dominant throughout the ripening process were Lactobacillus, Photobacterium, Leuconostoc, Weissella, and Lactococcus, while Carnobacterium, Brochothrix, and Acinetobacter were found also, but in negligible abundance. Among the culturable bacteria, Latilactobacillus sakei (Lactobacillus sakei) and Leuconostoc mesenteoides were present in all stages of ripening. © 2023 by the authors.

Structural modification of different carbon-based nanomaterials is often necessary toimprove their morphology and optical properties, particularly the incorporation of N-atoms ingraphene quantum dots (GQDs). Here, a clean, simple, one-step, and eco-friendly method forN-doping of GQDs using gamma irradiation is reported. GQDs were irradiated in the presenceof the different ethylenediamine (EDA) amounts (1 g, 5 g, and 10 g) and the highest % of N wasdetected in the presence of 10 g. N-doped GQDs emitted strong, blue photoluminescence (PL).Photoluminescence quantum yield was increased from 1.45, as obtained for non-irradiated dots,to 7.24% for those irradiated in the presence of 1 g of EDA. Modified GQDs were investigated asa PL probe for the detection of insecticide Carbofuran (2,2-Dimethyl-2,3-dihydro-1-benzofuran-7-yl methylcarbamate) and herbicide Amitrole (3-amino-1,2,4-triazole). The limit of detection was5.4 µmol L−1for Carbofuran. For the first time, Amitrole was detected by GQDs in a turn-off/turn-onmechanism using Pd(II) ions as a quenching agent. First, Pd(II) ions were quenched (turn-off) PLof GQDs, while after Amitrole addition, PL was recovered linearly with Amitrole concentration(turn-on). LOD was 2.03 µmol L−1. These results suggest that modified GQDs can be used as anefficient new material for Carbofuran and Amitrole detection. Furthermore, the phototoxicity of dotswas investigated on both Gram-positive and Gram-negative bacterial strains. When bacterial cellswere exposed to different GQD concentrations and illuminated with light of 470 nm wavelength, thetoxic effects were not observed.

INTRODUCTION Crown gall is an economically important and widespread plant disease caused by tumorigenic bacteria that are commonly affiliated with the genera Agrobacterium, Allorhizobium and Rhizobium. Novel and an atypical group of tumorigenic agrobacteria belonging to the genus Rhizobium (“tumorigenes” clade) was identified as a causative agent of crown/cane gall on blackberry, rhododendron and blueberry in Serbia and Germany (Kuzmanović et al., 2018 and 2019). Efficient measures to control crown gall disease were not reported till nowadays, so assessment and application of alternative biological control measures would contribute to sustainable agricultural production and environmental protection. The aims of the study were 1) identification of candidate bacterial strains that could be employed for biological control 2) to analyse phytobiome of the treated and non-treated crops and 3) to perform a whole-genome sequencing of a few most promising biocontrol strains. STATE OF THE ART Antimicrobial activity of ten biocontrol candidates from rhododendron and 27 additional antagonistic strains were tested in vitro against the tumor-inducing strain Rhizobium sp. rho-6.2. The six most efficient Pseudomonas and Bacillus strains were tested in vivo, using co-inoculation and preventive inoculation strategies in controlled greenhouse conditions on tomato plants as a model system in four replicas and randomized. Tumors from the most effective treatments were sampled, and then total DNA was isolated and subjected to the next-generation sequencing (NGS). Direct analysis of bacterial communities using Illumina MiSeq sequencing of 16S rRNA gene amplicon libraries was performed to assess the microbial ecological effect, with complete bioinformatic and computational biology analysis conducted. Also, a whole-genome sequencing of a few most promising antagonistic strains was performed. RESULTS Among six antagonistic strains, the most efficient in co-inoculation strategy against pathog

INTRODUCTION Maize is among the three world’s most important and widely grown cereals (Seyi-Amole & Onilude, 2021). The excessive and long-term application of agrochemicals for providing maize with essential nutrients, required for the development of all growth phenophases and for yield enhancement, leaves adverse consequences for human health and the environment (Khaliq et al., 2004). Although their use is inevitable to meet the increasing demand of the growing human population for a healthy food supply, organic fertilizers and biofertilizers (microbial fertilizers) are becoming recognized as effective, economically feasible, and environmentally sound alternatives for sustainable agriculture (Lawal & Babalola, 2014; Hui et al., 2017; Mahanty et al., 2017). The main objective of this study was to evaluate the influence of the microbial inoculant Phytobiotic (PHY), containing a consortium of Bacillus subtilis sp. subtilis and Microbacterium sp., on native maize microbiome during key growth phenophases (seedling, flowering, and harvesting) under field conditions, as well as to compare whether differences in efficacy between PHY, poultry manure (PM) and their combination (PHY_PM) exist, based on yield parameters. STATE OF THE ART Seeds, roots, and soil samples were taken for metabarcoding analysis during four growth phenophases (I-IV). Samples of uninoculated seeds and soil, poultry manure, and seeds inoculated with PHY were primarily taken before sowing (phenophase I). Further, during the growing season [phenophases II (seedling), III (flowering), and IV (harvesting)] the effect of PHY, PM, and PHY_PM on maize seeds, roots, and soil microbiome was evaluated in relation to concurrently sampled negative controls. A total DNA from the collected samples was isolated, amplified with primers 515F/ 806R targeting the V4 region of the 16S rRNA, and subjected to next-generation sequencing (NGS). The obtained sequence data were bioinformatically processed and used for the evalu

BACKGROUND Atypical Rhizobium sp. were identified as a causative agent of crown gall on rhododendron and blueberry in Germany. Considering the lack of efficient measures against crown gall disease, evaluation of innovative biocontrol measures would greatly contribute to sustainable agriculture. OBJECTIVES Identification of candidate bacterial strains that could be employed for biological control of the novel crown gall bacteria belonging to the ″tumorigenes″ clade. METHODS Antimicrobial activity of six antagonistic Pseudomonas and Bacillus strains were tested in vitro and in vivo against pathogenic strain Rhizobium sp. rho-6.2. The in vivo experiment, using co-inoculation and preventive inoculation strategies, was performed in controlled greenhouse conditions on tomato plants as a model system in four replicas and randomized. RESULTS The whole cultures of two antagonistic Pseudomonas strains were the most efficient against pathogenic Rhizobium sp. rho-6.2 in co-inoculation strategy with reducing tumor size of 92.86%. Contrary, in the preventive treatment same Pseudomonas strains were less efficient (15.38 and 30.77%). Despite the high in vitro antimicrobial activity of Bacillus strains their in vivo activity was less pronounced in preventive treatment (up to 15.38%), while in co-inoculation strategy was more prominent (42.86%). Consortium based on Bacillus and Pseudomonas strains, was increased biocontrol activity up to 38.6% of tumor’s reduction. The selected Pseudomonas strains could be further tested as an alternative strategy for the biocontrol of crown gall disease and potential involvement of quorum quenching mechanism will be determined.

The potential of two biocontrol strains, Bacillus velezensis X5-2 and Pseudomonas orientalis X2-1P to control black rot caused by Xanthomonas campestris pv. campestris was evaluated in vivo on autochthonous cabbage cultivar ‘Futoški’, under the condition of natural infection. Liquid formulations of biocontrol strains were prepared in fermenters (B. velezensis - 1010 CFU mL-1, P. orientalis - 109 CFU mL-1) and diluted with water in a 1:5 ratio, before use. Treatments were performed with each strain and their mixture (1:1). The experiment included the following: (i) seed treatments (15, 30, 60 min), (ii) foliar treatments (three treatments: when the first symptoms appeared and remaining every two weeks), and (iii) combined seed and foliar treatments. Experiments were conducted in four replicates, with 10 plants per replicate. Two controls were used, untreated and conventionally treated cabbage (pesticides). Assessment of disease intensity was rated one month after the last foliar treatment, using the Horsfall-Barratt scale (1-12). During ripening, cabbage heads’ weights were measured and data were statistically processed (Minitab). All performed treatments were effective in the control of black rot (85.7-98.9%). The highest efficacy was obtained in combined seed (60 min) and foliar treatments with P. orientalis, as well as its mixture with B. velezensis (98.9% both). All treatments influenced the increase of cabbage heads’ weight (up to 1.3 times), with the highest found in combined treatments (60 min seed + foliar) using a mixture of B. velezensis and P. orientalis. A negative correlation (P≤0.05) was found between disease intensity and cabbage heads’ weight.

Structural modification of different carbon-based nanomaterials is often necessary to improve their morphology and optical properties, particularly the incorporation of N-atoms in graphene quantum dots (GQDs). Here, a clean, simple, one-step, and eco-friendly method for N-doping of GQDs using gamma irradiation is reported. GQDs were irradiated in the presence of the different ethylenediamine (EDA) amounts (1 g, 5 g, and 10 g) and the highest % of N was detected in the presence of 10 g. N-doped GQDs emitted strong, blue photoluminescence (PL). Photoluminescence quantum yield was increased from 1.45, as obtained for non-irradiated dots, to 7.24% for those irradiated in the presence of 1 g of EDA. Modified GQDs were investigated as a PL probe for the detection of insecticide Carbofuran (2,2-Dimethyl-2,3-dihydro-1-benzofuran-7- yl methylcarbamate) and herbicide Amitrole (3-amino-1,2,4-triazole). The limit of detection was 5.4 µmol L−1 for Carbofuran. For the first time, Amitrole was detected by GQDs in a turn-off/turn-on mechanism using Pd(II) ions as a quenching agent. First, Pd(II) ions were quenched (turn-off) PL of GQDs, while after Amitrole addition, PL was recovered linearly with Amitrole concentration (turn-on). LOD was 2.03 µmol L−1 . These results suggest that modified GQDs can be used as an efficient new material for Carbofuran and Amitrole detection. Furthermore, the phototoxicity of dots was investigated on both Gram-positive and Gram-negative bacterial strains. When bacterial cells were exposed to different GQD concentrations and illuminated with light of 470 nm wavelength, the toxic effects were not observed.

Maize (Zea mays L.) occupies a significant part of the world cultivated area. Beneficial microorganisms and organic fertilizers are powerful tools for sustainable agricultural management. Applying appropriate formulation either through seed coating or composting material together with seeds during the sowing was aimed to achieve optimal performance on plants and increase the crop yields. Also, a complete analysis of phytobiome including high-throughput amplicon and whole-genome sequencing, and computational biology of the treated and non-treated plants' roots and soil was applied. The statistically significant difference in bacterial communities between phenophases and treatments was much more prevalent in the roots than soil itself. In general, seedling phenophase (II) differed in terms of diversity from bacterial communities of flowering (III) and harvesting phenophases (IV), regardless of treatment, indicating good colonization with the treated substrate for up to one month. In the root samples, the dominance of the genus Pseudomonas was evident, in the initial stages (phase II), while this percentage decreased linearly over time. High relative abundance of the genera Achromobacter, Sphingobacterium, and Stenotrophomonas also characterized II phenophase. Contrary, a relative abundance of the genus Bacillus was characteristic for III phenophase, and especially twice or three times higher in treatments with phytobiotic or manure compared to the negative control. Furthermore, it was observed that with increasing inoculation of Bacillus isolate, the percentage of Pantoea genus decreases drastically in coated seeds. In conclusion, a statistically significant increase in maize yield was obtained in the treatment with phytobiotic with an average value of 650 kg/ha compared to the negative control.

Plant pathogenic bacteria from the genera Pectobacterium and Dickeya in association with potato cause blackleg and tuber soft rot disease. A metabarcoding was used to analyze the microbial communities of soft-rotted tubers and their geocaulospheres. In September 2018, samples collected from the blackleg-infected feld (58 ha) in the Bačka region (Serbia) reached 45% disease incidence. Next-generation sequencing Illumina MiSeq platform was used to obtain 16S rRNA sequences from six tested tubers (with and without soft rot symptom) and the surrounding geocaulosphere. Proteobacteria, Bacteroidetes, Firmicutes, and Actinobacteria were present in all samples, with Proteobacteria being the most prevalent, especially in tubers without soft rot symptoms. In all tested samples, species of the Pectobacterium (P. aroidearum, P. atrosepticum, P. carotovorum, and P. polaris) were detected but were more represented in tubers without symptoms. However, the genus Dickeya was at the limit of detection or not detected at all. Acinetobacter was the most dominant in tubers with soft rot, while Pseudomonas and Enterobacter were the most abun dant in tubers without symptoms. The genera Bacteroides and Dysgonomonas with starch-degradable features were almost exclusively present in soft rotted tubers and their corresponding geocaulospheres. The most represented genera in the geocaulosphere sample associated with no-symptom tubers were Gaiella, Sphingomonas, Sphingobium, Gemmatimonas, and Geminicoccus, which include species with confrmed biocontrol potential. This study indicates that the soft rot maceration process arises due to complex interactions between plant pathogens and other endophytic bacteria.

During a survey of diatoms in the artificial sandpit lake “Peskara” in the protected zone of the Rusanda Nature Park, a large number of brackish taxa at epipsamic and epiphytic communities were recorded (e.g. Bacillaria paxillifera, Caloneis permagna, Ctenophora pulchella, Entomoneis costata. Haslea duerrenbergiana, Navicula salinarum, Nitzschia agnita, N. filiformis, Staurophora brantii, Seminavis strigosa, Tryblionella apiculata). One new genus Seminavis and 15 diatom species were noted for the first time for the diatom flora of Serbia. Dominant taxa changed over seasons without any regularity. In spring 2021, we have observed an increased abundance of centric species Thalassiosira pseudonana and Stephanodiscus parvus while in other seasons it was difficult to discern a pattern of dominance among benthic diatoms. Metabarcoding analysis of the same sample using 18S rRNA as a gene marker confirmed the dominance of centric diatoms. This study contributes to a better understanding of brackish diatom taxa found in inland habitats, many of which can be utilized as useful tools for the ecological status assessment.

Structural modification of different carbon-based nanomaterials is often necessary toimprove their morphology and optical properties, particularly the incorporation of N-atoms ingraphene quantum dots (GQDs). Here, a clean, simple, one-step, and eco-friendly method forN-doping of GQDs using gamma irradiation is reported. GQDs were irradiated in the presenceof the different ethylenediamine (EDA) amounts (1 g, 5 g, and 10 g) and the highest % of N wasdetected in the presence of 10 g. N-doped GQDs emitted strong, blue photoluminescence (PL).Photoluminescence quantum yield was increased from 1.45, as obtained for non-irradiated dots,to 7.24% for those irradiated in the presence of 1 g of EDA. Modified GQDs were investigated asa PL probe for the detection of insecticide Carbofuran (2,2-Dimethyl-2,3-dihydro-1-benzofuran-7-yl methylcarbamate) and herbicide Amitrole (3-amino-1,2,4-triazole). The limit of detection was5.4 µmol L−1for Carbofuran. For the first time, Amitrole was detected by GQDs in a turn-off/turn-onmechanism using Pd(II) ions as a quenching agent. First, Pd(II) ions were quenched (turn-off) PLof GQDs, while after Amitrole addition, PL was recovered linearly with Amitrole concentration(turn-on). LOD was 2.03 µmol L−1. These results suggest that modified GQDs can be used as anefficient new material for Carbofuran and Amitrole detection. Furthermore, the phototoxicity of dotswas investigated on both Gram-positive and Gram-negative bacterial strains. When bacterial cellswere exposed to different GQD concentrations and illuminated with light of 470 nm wavelength, thetoxic effects were not observed.

The olive tree is one of the most important agricultural plants, affected by several pests and diseases that cause a severe decline in health status leading to crop losses. Olive leaf spot disease caused by the fungus Venturia oleaginea can result in complete tree defoliation and consequently lower yield. The aim of the study was to obtain new knowledge related to plant–pathogen interaction, reveal mechanisms of plant defense against the pathogen, and characterize fungal phyllosphere communities on infected and symptomless leaves that could contribute to the development of new plant breeding strategies and identification of novel biocontrol agents. The highly susceptible olive variety “Istrska Belica”' was selected for a detailed evaluation. Microscopy analyses led to the observation of raphides in the mesophyll and parenchyma cells of infected leaves and gave new insight into the complex V. oleaginea pathogenesis. Culturable and total phyllosphere mycobiota, obtained via metabarcoding approach, highlighted Didymella, Aureobasidium, Cladosporium, and Alternaria species as overlapping between infected and symptomless leaves. Only Venturia and Erythrobasidium in infected and Cladosporium in symptomless samples with higher abundance showed statistically significant differences. Based on the ecological role of identified taxa, it can be suggested that Cladosporium species might have potential antagonistic effects on V. oleaginea. © 2022, The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.

Utvrđivanje potencijalnih korelacija, između fenotipskih karakteristika (osetljivost na različite klase antibiotika, ekspresija različitih klasa β-laktamaza, učestalost serotipova, proizvodnja pigmenata i stvaranje biofilma) i genotipskih obrazaca kliničkih izolata Pseudomonas aeruginosa različitog porekla, predstavljalo je glavni cilj ovog rada. Tokom dve faze istraživanja, ispitano je 147 kliničkih izolata, od kojih je najveći procenat dobijen iz briseva rana (66%) i urino-kultura (10,9%). Polivalentne serogrupe PMA i PMF, kao i monovalentni serotipovi P1, P6 i P11 bili su najzastupljeniji, dok je najveći procenat izolata bio očekivano netipabilan. Istovremena produkcija oba pigmenta karakterisala je najveći broj izolata, dok su oni sa serotipovima P1, P6 i P11 produkovali više pioverdina nego piocijanina. Sposobnost formiranja biofilma imalo je 77,66% kliničkih izolata, a najizraženija je utvrđena za izolate iz urinokultura i sputuma. Najveći stepen osetljivosti utvrđen je za kolistin, aztreonam, karbapeneme i piperacilin/tazobaktam, dok je izraženija otpornost prikazana prema aminoglikozidima i fluorohinolonima. Višestruku otpornost na tri ili više klasa antibiotika je pokazalo 30,9% izolata iz druge faze istraživanjaje, a skoro polovina je eksprimirala β-laktamaze proširenog spektra, karbapenemaze i metalo-β-laktamaze. RAPD272 PCR analiza pokazala se kao najbolja tehnika koja je izdvojila najveći broj klastera sa ujednačenim obrascima, ističući izraženu heterogenost izolata iste vrste. Korelacija između genetičkih obrazaca i fenotipskih karakteristika nije ustanovljena, ukazujući na potrebu za novim molekularnim metodama u ovom delu Srbije u cilju otkrivanja potencijalnih veza među njima i na izraženu rezistenciju koja se poslednjih godina uvećava., Determining potential correlations between phenotypic characteristics (sensitivity to different classes of antibiotics, expression of different β-lactamases classes, frequency of serotypes, pigment and biofilm production) and genotypic patterns of clinical isolates of Pseudomonas aeruginosa from different origins was the main goal of this study. Study itself was splitted into two phases, a total of 147 clinical isolates were examined, out of which the largest percentage were from wound swabs (66%) and urine cultures (10.9%). Polyvalent serogroups PMA and PMF, as well as monovalent serotypes P1, P6 and P11 were the most represented, while the highest percentage of isolates was atypical. Simultaneous production of both pigments characterized the most of isolates, while those with serotypes P1, P6 and P11 produced more pyoverdine than pyocyanin. Even 77.66% of clinical isolates had the ability to form a biofilm, and the most pronounced was found for isolates from urine cultures and sputum. The highest susceptibility was found for colistin, aztreonam, carbapenems and piperacillin/tazobactam, while more pronounced resistance was shown onto aminoglycosides and fluoroquinolones. Multiple resistances, up to three or more antibiotics classes, were shown by 30.9% of isolates from the second study phase, and almost half of them expressed broad-spectrum β-lactamases, carbapenemases and metallo-β-lactamases. RAPD272 PCR analysis proved to be the best technique that singled out the largest number of clusters with uniform patterns, emphasizing the pronounced heterogeneity of isolates of the same species. The correlation between genetic patterns and phenotypic characteristics has not been established, indicating the need for new molecular methods in this part of Serbia to discover potential links between them and on the pronounced resistance, which has increased in recent years.

Sa namerom definisanja hidrogeološke uloge i značaja mikrobiološkog diverziteta odabranih pojava mineralnih voda Srbije, primenom sistema biodetektora, dokazano je prisustvo gvožđevitih, sulfato-redukujućih, sluz-produkujućih, heterotrofnih aerobnih i denitrifikacionih bakterija, na osnovu čega je proračunat rizik od razvoja procesa biokorozije i biohemijske inkrustacije, te zdravstveni rizik. Uz procenu rizika, izvedene su analize skenirajuće elektronske mikroskopije biofilmova i hidrogeohemijsko modeliranje, te makroskopska, otpička i difrakciona ispitivanja naslaga inkrustacije, dok je fizičko-hemijskim analizama utvrđena vrednost ukupne mineralizacije iznad 1 g/L, kao i povišeni sadržaji odgovarajućih metala i nemetala. Metabarkoding analizama mikrobioma mineralnih voda dokazano je prisustvo razdela Proteobacteria, Bacteroidetes, Patescibacteria, Chlorflexi, Firmicutes, Spirochaetes i Acidobacteria u većem procentu. Pseudomonas, Limnobacter i Rheinheimera su bili najzastupljeniji rodovi u uzorku mineralnih voda Torde, Acinetobacter i Massilia u uzorku iz Obrenovačke Banje, Maritimimonas, Phreatobacter, Planktosalinus, Acinetobacter, Geminocystis i Sulfuritalea u uzorku iz Lomnice, Pseudorhodobacter i Limnohabitans u mineralnim vodama Velike Vrbnice, dok su u uzorku iz Slankamen Banje dominirale zelene sumporne bakterije, Methylotenera, Flavobacterium, Melioribacter i Dechloromonas. Realizovanim istraživanjima zaključeno je da odgovarajući predstavnici mikroorganizama utiču na starenje bunara i definišu ekološki status vodnih resursa, dok s druge strane pojedini rodovi poseduju biotehnološki potencijal u tretmanu remedijacije kontaminiranih voda, zbog čega postoji neophodnost implementacije biohidrogeologije u okviru standardne metodike hidrogeoloških istraživanja., The presence of iron related, sulphate reducing, slime producing, heterotrophic aerobic and denitrifying bacteria was proved applying biodetectors, in order to define the hydrogeological role and importance of the microbiological diversity of select occurrences of mineral waters in Serbia. Then the biocorrosion, biochemical encrustation and health risks were assessed. The research included scanning electron microscopy of biofilms, hydrogeochemical modelling and macroscopic, optical and diffraction examination of the encrustations. Physical and chemical analyses indicated total dissolved salts of more than 1 g/L and elevated concentrations of certain metals and non-metals. Metabarcoding of the microbiomes of the mineral waters revealed the presence of a high percentage of the Proteobacteria, Bacteroidetes, Patescibacteria, Chlorflexi, Firmicutes, Spirochaetes and Acidobacteria divisions. Pseudomonas, Limnobacter and Rheinheimera were the most representative genera in the mineral water sample from Torda, Acinetobacter and Massilia in the sample collected in Obrenovačka Banja, Maritimimonas, Phreatobacter, Planktosalinus, Acinetobacter, Geminocystis and Sulfuritalea in the sample from Lomnica, and Pseudorhodobacter and Limnohabitans in the sample from Velika Vrbnica, while in the sample from Slankamen Banja was dominated by green sulphur bacteria, Methylotenera, Flavobacterium, Melioribacter and Dechloromonas. The research concludes that the respective microorganisms cause water well aging and define the ecological status of the water resources, while, on the other hand, some of the genera possess the biotechnological potential for treating and remediating contaminated waters, which is a compelling reason for implementing biohydrogeology in the standard hydrogeological research methodology

Antibacterial and antibiofilm efficacy through a synergistic effect of herbal leaf extracts of bearberry (BE), bearberry tea (BTE), parsley (PE), and the strawberry tree (STE) was evaluated to discover an alternative approach to control urinary tract infections caused by Enterococci. UHPLC–DAD MS/MS analysis indicated that herbal extracts were rich in ellagic and gallic acids, catechin, quercetin, and arbutin, which are strong antioxidants. Mostly, Enterococcus faecalis isolates showed resistance only to gentamicin and/or norfloxacin (26.3 %) and moderate biofilm production (31.5 %), while 12 isolates of Enterococcus faecium showed multiple antibiotic resistance. The minimum inhibitory concentration for the majority of isolates was 1.25 mg ml−1 for STE, but 2.5 mg ml−1 for BE and BTE. Although individual extracts gave higher MIC values, for most isolates, a synergistic effect at lower concentrations was achieved, when BE or BTE (0.625 mg ml−1) was combined with STE (0.31 mg ml−1). The BTE extract was shown to have the strongest individual effect on initial adhesiveness and on biofilm formation to all selected isolates, while the synergism of BTE and STE caused significant biofilm inhibition at all concentrations tested. The synergistic effect achieved of the tested extract combinations might be a starting point in the development of alternative products, effective against antibiotic resistant Enterococcus isolates.

Antibacterial and antibiofilm efficacy through a synergistic effect of herbal leaf extracts of bearberry (BE), bearberry tea (BTE), parsley (PE), and the strawberry tree (STE) was evaluated to discover an alternative approach to control urinary tract infections caused by Enterococci. UHPLC–DAD MS/MS analysis indicated that herbal extracts were rich in ellagic and gallic acids, catechin, quercetin, and arbutin, which are strong antioxidants. Mostly, Enterococcus faecalis isolates showed resistance only to gentamicin and/or norfloxacin (26.3 %) and moderate biofilm production (31.5 %), while 12 isolates of Enterococcus faecium showed multiple antibiotic resistance. The minimum inhibitory concentration for the majority of isolates was 1.25 mg ml−1 for STE, but 2.5 mg ml−1 for BE and BTE. Although individual extracts gave higher MIC values, for most isolates, a synergistic effect at lower concentrations was achieved, when BE or BTE (0.625 mg ml−1) was combined with STE (0.31 mg ml−1). The BTE extract was shown to have the strongest individual effect on initial adhesiveness and on biofilm formation to all selected isolates, while the synergism of BTE and STE caused significant biofilm inhibition at all concentrations tested. The synergistic effect achieved of the tested extract combinations might be a starting point in the development of alternative products, effective against antibiotic resistant Enterococcus isolates.

Plant pathogenic bacterium Xanthomonas campestris pv. campestris (Xcc) was recently described as a pathogen of winter oilseed rape in Serbia. A metabarcoding approach was used to study bacterial community composition changes in the phyllosphere and rhizosphere of different winter oilseed rape types (lines, cultivars, and hybrids) naturally infected with Xcc. This study also aimed to examine the potential of indigenous Bacillus and Pseudomonas isolates to suppress Xcc. Infection with Xcc leads to depletion of bacterial diversity in the plants' phyllosphere and explicitly decreasing the abundance of genera such as Exiguobacterium, Massilia, and Pantoea with potentially beneficial properties. Strains identified as Bacillus velezensis X5-2, Bacillus megaterium X6-3, and Pseudomonas orientalis X2-1P obtained from oilseed rape phyllosphere were found to be effective against Xcc in vitro and in vivo when applied as a whole-culture and as a cell-free supernatant. The greenhouse in vivo tests on winter oilseed rape plants with three selected biocontrol strains lead to a disease reduction of 82.37% and 72.47% in preventive and curative treatments, respectively. Genetic screening showed potential for the biosynthesis of surfactin, kurstakin, bacillomycin D, and iturin in B. velezensis X5-2, as well as surfactin and kurstakin in B. megaterium X6-3. Gene phcA encoding phenazine-1-carboxylic acid was detected in P. orientalis X2-1P. The chemical composition of ethyl acetate and benzene extracts of three biocontrol strains obtained by GC–MS and HPLC-ESI-qTOF/MS analyses indicates numerous volatile organic compounds (alkenes, benzenes, carboxylic acids, indoles, pyrazines, etc.), lipopeptides, and/or antibiotics, for many of which antimicrobial potential is proven. We assume that this wide range of metabolites is responsible for the exhibited biocontrol activity against Xcc. These results recommend all three biocontrol strains for further studies for use as agents for biocontrol of

Sa namerom definisanja hidrogeološke uloge i značaja mikrobiološkog diverziteta odabranih pojava mineralnih voda Srbije, primenom sistema biodetektora, dokazano je prisustvo gvožđevitih, sulfato-redukujućih, sluz-produkujućih, heterotrofnih aerobnih i denitrifikacionih bakterija, na osnovu čega je proračunat rizik od razvoja procesa biokorozije i biohemijske inkrustacije, te zdravstveni rizik. Uz procenu rizika, izvedene su analize skenirajuće elektronske mikroskopije biofilmova i hidrogeohemijsko modeliranje, te makroskopska, otpička i difrakciona ispitivanja naslaga inkrustacije, dok je fizičko-hemijskim analizama utvrđena vrednost ukupne mineralizacije iznad 1 g/L, kao i povišeni sadržaji odgovarajućih metala i nemetala. Metabarkoding analizama mikrobioma mineralnih voda dokazano je prisustvo razdela Proteobacteria, Bacteroidetes, Patescibacteria, Chlorflexi, Firmicutes, Spirochaetes i Acidobacteria u većem procentu. Pseudomonas, Limnobacter i Rheinheimera su bili najzastupljeniji rodovi u uzorku mineralnih voda Torde, Acinetobacter i Massilia u uzorku iz Obrenovačke Banje, Maritimimonas, Phreatobacter, Planktosalinus, Acinetobacter, Geminocystis i Sulfuritalea u uzorku iz Lomnice, Pseudorhodobacter i Limnohabitans u mineralnim vodama Velike Vrbnice, dok su u uzorku iz Slankamen Banje dominirale zelene sumporne bakterije, Methylotenera, Flavobacterium, Melioribacter i Dechloromonas. Realizovanim istraživanjima zaključeno je da odgovarajući predstavnici mikroorganizama utiču na starenje bunara i definišu ekološki status vodnih resursa, dok s druge strane pojedini rodovi poseduju biotehnološki potencijal u tretmanu remedijacije kontaminiranih voda, zbog čega postoji neophodnost implementacije biohidrogeologije u okviru standardne metodike hidrogeoloških istraživanja., The presence of iron related, sulphate reducing, slime producing, heterotrophic aerobic and denitrifying bacteria was proved applying biodetectors, in order to define the hydrogeological role and importance of the microbiological diversity of select occurrences of mineral waters in Serbia. Then the biocorrosion, biochemical encrustation and health risks were assessed. The research included scanning electron microscopy of biofilms, hydrogeochemical modelling and macroscopic, optical and diffraction examination of the encrustations. Physical and chemical analyses indicated total dissolved salts of more than 1 g/L and elevated concentrations of certain metals and non-metals. Metabarcoding of the microbiomes of the mineral waters revealed the presence of a high percentage of the Proteobacteria, Bacteroidetes, Patescibacteria, Chlorflexi, Firmicutes, Spirochaetes and Acidobacteria divisions. Pseudomonas, Limnobacter and Rheinheimera were the most representative genera in the mineral water sample from Torda, Acinetobacter and Massilia in the sample collected in Obrenovačka Banja, Maritimimonas, Phreatobacter, Planktosalinus, Acinetobacter, Geminocystis and Sulfuritalea in the sample from Lomnica, and Pseudorhodobacter and Limnohabitans in the sample from Velika Vrbnica, while in the sample from Slankamen Banja was dominated by green sulphur bacteria, Methylotenera, Flavobacterium, Melioribacter and Dechloromonas. The research concludes that the respective microorganisms cause water well aging and define the ecological status of the water resources, while, on the other hand, some of the genera possess the biotechnological potential for treating and remediating contaminated waters, which is a compelling reason for implementing biohydrogeology in the standard hydrogeological research methodology

Antibacterial and antibiofilm efficacy through a synergistic effect of herbal leaf extracts of bearberry (BE), bearberry tea (BTE), parsley (PE), and the strawberry tree (STE) was evaluated to discover an alternative approach to control urinary tract infections caused by Enterococci. UHPLC–DAD MS/MS analysis indicated that herbal extracts were rich in ellagic and gallic acids, catechin, quercetin, and arbutin, which are strong antioxidants. Mostly, Enterococcus faecalis isolates showed resistance only to gentamicin and/or norfloxacin (26.3 %) and moderate biofilm production (31.5 %), while 12 isolates of Enterococcus faecium showed multiple antibiotic resistance. The minimum inhibitory concentration for the majority of isolates was 1.25 mg ml−1 for STE, but 2.5 mg ml−1 for BE and BTE. Although individual extracts gave higher MIC values, for most isolates, a synergistic effect at lower concentrations was achieved, when BE or BTE (0.625 mg ml−1) was combined with STE (0.31 mg ml−1). The BTE extract was shown to have the strongest individual effect on initial adhesiveness and on biofilm formation to all selected isolates, while the synergism of BTE and STE caused significant biofilm inhibition at all concentrations tested. The synergistic effect achieved of the tested extract combinations might be a starting point in the development of alternative products, effective against antibiotic resistant Enterococcus isolates.

Winter oilseed rape has recently been described as a new host for the phytopathogenic bacteriumXanthomonas campestrispv.campestris(Xcc) in Serbia. In order to provide information about the genetic structure of this bacterium's population and its pathogenic features on this crop, a collection of 65 winter oilseed rape Xcc isolates obtained over a 5-year period (2014-2018) from different Serbian localities was examined. The presence of intrapathovar diversity within the tested isolates was confirmed by repetitive element PCR (rep-PCR), revealing 14, 10, and 4 different profiles for BOX, ERIC, and REP primers, respectively. A multilocus sequence typing (MLST) approach separated the isolates into seven groups on a phylogenetic tree. The PubMLST non-redundant database was used to compare sequences of four housekeeping genes from isolates with 75 strains ofXanthomonasspp. to determine nucleotide changes caused by host-pathogen coevolution. Five allelic profile combinations (ST3, ST5, ST9, ST26, and ST47) were determined, with ST47 being the most frequent. Pathogenicity and host range tests showed higher virulence of the tested isolates on winter oilseed rape than on otherBrassica oleraceahosts (broccoli, cabbage, cauliflower, collard greens, kale, and kohlrabi). The most aggressive isolates were NCPPB4679, NCPPB4680, and Xc361, belonging to genotype ST26. Fifty oilseed rape lines, cultivars, and hybrids showed susceptibility to the three Xcc isolates tested (of high, low, and moderate virulence), indicating that there was no resistance to the bacterium. The results provide significant information about diversification of Xcc isolates and their evolution toward specialization for winter oilseed rape.

Plant pathogenic bacterium Xanthomonas campestris pv. campestris (Xcc) was recently described as a pathogen of winter oilseed rape in Serbia. A metabarcoding approach was used to study bacterial community composition changes in the phyllosphere and rhizosphere of different winter oilseed rape types (lines, cultivars, and hybrids) naturally infected with Xcc. This study also aimed to examine the potential of indigenous Bacillus and Pseudomonas isolates to suppress Xcc. Infection with Xcc leads to depletion of bacterial diversity in the plants' phyllosphere and explicitly decreasing the abundance of genera such as Exiguobacterium, Massilia, and Pantoea with potentially beneficial properties. Strains identified as Bacillus velezensis X5-2, Bacillus megaterium X6-3, and Pseudomonas orientalis X2-1P obtained from oilseed rape phyllosphere were found to be effective against Xcc in vitro and in vivo when applied as a whole-culture and as a cell-free supernatant. The greenhouse in vivo tests on winter oilseed rape plants with three selected biocontrol strains lead to a disease reduction of 82.37% and 72.47% in preventive and curative treatments, respectively. Genetic screening showed potential for the biosynthesis of surfactin, kurstakin, bacillomycin D, and iturin in B. velezensis X5-2, as well as surfactin and kurstakin in B. megaterium X6-3. Gene phcA encoding phenazine-1-carboxylic acid was detected in P. orientalis X2-1P. The chemical composition of ethyl acetate and benzene extracts of three biocontrol strains obtained by GC-MS and HPLC-ESI-qTOF/MS analyses indicates numerous volatile organic compounds (alkenes, benzenes, carboxylic acids, indoles, pyrazines, etc.), lipopeptides, and/or antibiotics, for many of which antimicrobial potential is proven. We assume that this wide range of metabolites is responsible for the exhibited biocontrol activity against Xcc. These results recommend all three biocontrol strains for further studies for use as agents for biocontrol of

Winter oilseed rape has recently been described as a new host for the phytopathogenic bacteriumXanthomonas campestrispv.campestris(Xcc) in Serbia. In order to provide information about the genetic structure of this bacterium's population and its pathogenic features on this crop, a collection of 65 winter oilseed rape Xcc isolates obtained over a 5-year period (2014-2018) from different Serbian localities was examined. The presence of intrapathovar diversity within the tested isolates was confirmed by repetitive element PCR (rep-PCR), revealing 14, 10, and 4 different profiles for BOX, ERIC, and REP primers, respectively. A multilocus sequence typing (MLST) approach separated the isolates into seven groups on a phylogenetic tree. The PubMLST non-redundant database was used to compare sequences of four housekeeping genes from isolates with 75 strains ofXanthomonasspp. to determine nucleotide changes caused by host-pathogen coevolution. Five allelic profile combinations (ST3, ST5, ST9, ST26, and ST47) were determined, with ST47 being the most frequent. Pathogenicity and host range tests showed higher virulence of the tested isolates on winter oilseed rape than on otherBrassica oleraceahosts (broccoli, cabbage, cauliflower, collard greens, kale, and kohlrabi). The most aggressive isolates were NCPPB4679, NCPPB4680, and Xc361, belonging to genotype ST26. Fifty oilseed rape lines, cultivars, and hybrids showed susceptibility to the three Xcc isolates tested (of high, low, and moderate virulence), indicating that there was no resistance to the bacterium. The results provide significant information about diversification of Xcc isolates and their evolution toward specialization for winter oilseed rape.

The main focus of this study is to assess radical scavenging and antimicrobial activities of the 11 wood extracts: oak (Quercus petraea (Matt.) Liebl., Q. robur L., and Q. cerris L.), mulberry (Morus alba L.), myrobalan plum (Prunus cerasifera Ehrh.), black locust (Robinia pseudoacacia L.), and wild cherry (Prunus avium L.). High-performance thin-layer chromatography (HPTLC) provided initial phenolic screening and revealed different chemical patterns among investigated wood extracts. To identify individual compounds with radical scavenging activity DPPH-HPTLC, assay was applied. Gallic acid, ferulic and/or caffeic acids were identified as the compounds with the highest contribution of total radical scavenging activity. Principal component analysis was applied on the data set obtained from HPTLC chromatogram to classify samples based on chemical fingerprints: Quercus spp. formed separate clusters from the other wood samples. The wood extracts were evaluated for their antimicrobial activity against eight representative human and opportunistic pathogens. The lowest minimum inhibitory concentration (MIC) was recorded against Staphylococcus aureus for black locust, cherry and mulberry wood extracts. This work provided simple, low-cost and high-throughput screening of phenolic compounds and assessments of the radical scavenging properties of selected individual metabolites from natural matrix that contributed to scavenge free radicals.

The Allchar mineral mine is one of the oldest arsenic-antimony mines in the Republic of North Macedonia. The mine is a well-known reservoir of the worldwide purest source of the thallium-bearing mineral, lorandite (TlAsS2). The current study evaluated the bacterial and fungal diversity of three As- and Tl-contaminated sites in Allchar mineral mine. We used a combination of high-throughput sequencing and bioinformatic analyses. Trace metal content was detected using inductively coupled plasma optical emission spectrometry. Our analysis showed the presence of 25 elements and confirmed a high concentration of As and Tl. Alpha diversity indices suggested a high diversity and evenness of bacterial and fungal communities. Bacterial phyla that dominated the environment were Bacteroidetes, Acidobacteria, Planctomycetes, Actinobacteria and Verrucomicrobia. Looking at the genus level, we found the following groups of bacteria: Chryseolinea, Opitutus, Flavobacterium, Pseudomonas, Terrimonas, Sphingomonas and Reyranella. For the fungi genera, we report Tetracladium sp., Coprinellus micaceus, Coprinus sp. from Ascomycota and Basidiomycota phyla in all sites. We also observed a high abundance of the fungal species Pilidium sp., Dendroclathra lignicola, Rosellinia desmazieri, Hypomyces rosellus and Coprinellus disseminatus. This study is the first to identify specific As- and Tl-tolerant fungal (Pilidium sp., Cladophialophora sp., Neobulgaria sp. and Mycena acicula) and bacterial (Trichococcus, Devosia, Litorilinea and Gimesia) genera from Allchar mine, suggesting bioremediation and industrial potential. © 2020 Oxford University Press. All rights reserved.

Ethnobotanical and ethnopharmacological studies of many Centaurea species indicated their potential in folk medicine so far. However, investigations of different Centaurea calcitrapa L. extracts in terms of cytotoxicity and antimicrobial activity against phytopathogens are generally scarce. The phenolic profile and broad antimicrobial activity (especially towards bacterial phytopathogens) of methanol (MeOH), 70% ethanol (EtOH), ethyl-acetate (EtOAc), 50% acetone (Me2CO) and dichloromethane: methanol (DCM: MeOH, 1: 1) extracts of C. calcitrapa leaves and their potential toxicity on MRC-5 cell line were investigated for the first time. A total of 55 phenolic compounds were identified: 30 phenolic acids and their derivatives, 25 flavonoid glycosides and aglycones. This is also the first report of the presence of centaureidin, jaceidin, kaempferide, nepetin, flavonoid glycosides, phenolic acids and their esters in C. calcitrapa extracts. The best results were obtained with EtOAc extract with lowest MIC values expressed in µg/mL ranging from 13 to 25, while methicillin resistant Staphylococcus aureus was the most susceptible strain. The most susceptible phytopathogens were Pseudomonas syringae pv. syringae, Xanthomonas campestris pv. campestris and Agrobacterium tumefaciens. The highest cytotoxicity was recorded for EtOAc and Me2CO extracts with the lowest relative and absolute IC50 values between 88 and 102 µg/mL, while EtOH extract was the least toxic with predicted relative IC50 value of 1578 µg/mL. Our results indicate that all tested extracts at concentration considered as non-toxic can be one of great importance in combat towards phytopathogenic and human pathogenic strains, as well as natural sources of antimicrobials.

Linden tea has been used in Serbian folk medicine for centuries to induce sweating for colds, relieve throat irritation and cough, reduce blood pressure, as well as a diuretic, spasmolytic, and as a sedative. The main aim of this study was to determine the comprehensive metabolite profile, free radical scavenging activity, and antimicrobial activities against 23 human and plant pathogens of both commercial and field-collected linden tea samples from Serbia. Developed high-performance thin-layer chromatography (HPTLC) method allowed identification of eight major metabolites in investigated samples, while forty-six metabolites were tentatively identified using high resolution mass spectrometry (HRMS): 14 phenolic acids and their derivatives, 15 glycosides, 9 flavonoids, and 8 procyanidins. Both commercial and field-collected linden tea samples showed similar total phenolic content and radical scavenging activity. Seven compounds such as p-hydroxybenzoic acid 4-O-hexoside, caffeic acid cinnamyl ester, pinocembrin, galangin, luteolin 7-O-glucuronide (and its isomer), isorhamnetin hexosyl hexoside were found in Tilia samples for the first time. Radical reagent 2,2-Diphenyl-1-picrylhydrazyl· (DPPH·) was used in DPPH-HPTLC assay which identified chlorogenic acid, caffeic acid, astragalin, quercitrin, tiliroside, and rutin as compounds that exhibit radical scavenging activity. Tilia samples showed inhibitory effect overwhelming only on Gram-positive bacteria, especially on Bacillus subtilis with the lowest MIC values observed, as well as towards Staphylococcus aureus and oral cavity isolates of Streptococcus pyogenes and Streptococcus mutans. Vaginal isolate of Candida glabrata showed more susceptibility than Candida albicans isolate. Generally, extract from Tilia cordata Miller (L4) showed the highest antimicrobial activity against the most of the tested pathogens, among all field-collected Tilia samples.

Aims: A multidisciplinary approach was used to compare phenolic composition, radical scavenging and antimicrobial activity of propolis samples from different geographical localities, and plant resin against various microorganisms. Methods and Results: Using UHPLC-qqqMS quantitative analysis, 28 phenolic compounds were determined. Caffeic and p-coumaric acids were identified as main phenolic acids in poplar propolis samples, except samples from Russia (P6) and China (P7). Radical scavenging activity (applying DPPH spectrophotometric assay) showed the highest activity of Serbian (40·51%) and Chinese (53·21%) propolis samples. Broth microdilution method was used for the oral cavity, fungal phytopathogenic and human vaginal isolates which have been identified at a molecular level. The most sensitive bacterial isolates were Lactobacillus acidophilus (MIC of 0·03–0·13 mg ml−1) and the oral streptococci isolates (MIC values of 0·19–0·13 mg ml−1). The most sensitive fungal phytopathogenic isolate was Fusarium oxysporum (MIC 0·003 mg ml−1). All samples, except propolis from Serbia (P4) and Turkey (P5), showed a strong antifungal activity against Fusarium sporotrichioides, Fusarium subglutinans and Fusarium proliferatum. Conclusion: The results of various tests indicate good radical scavenging and antimicrobial activity against important human and plant pathogens. Significance and Impact of the Study: A detailed propolis analysis is important when proposing a preparation of new biological antimicrobial products which have a positive impact on human health and reduce antibacterial resistance.

Ethnobotanical and ethnopharmacological studies of many Centaurea species indicated their potential in folk medicine so far. However, investigations of different Centaurea calcitrapa L. extracts in terms of cytotoxicity and antimicrobial activity against phytopathogens are generally scarce. The phenolic profile and broad antimicrobial activity (especially towards bacterial phytopathogens) of methanol (MeOH), 70% ethanol (EtOH), ethyl-acetate (EtOAc), 50% acetone (Me2CO) and dichloromethane: methanol (DCM: MeOH, 1: 1) extracts of C. calcitrapa leaves and their potential toxicity on MRC-5 cell line were investigated for the first time. A total of 55 phenolic compounds were identified: 30 phenolic acids and their derivatives, 25 flavonoid glycosides and aglycones. This is also the first report of the presence of centaureidin, jaceidin, kaempferide, nepetin, flavonoid glycosides, phenolic acids and their esters in C. calcitrapa extracts. The best results were obtained with EtOAc extract with lowest MIC values expressed in µg/mL ranging from 13 to 25, while methicillin resistant Staphylococcus aureus was the most susceptible strain. The most susceptible phytopathogens were Pseudomonas syringae pv. syringae, Xanthomonas campestris pv. campestris and Agrobacterium tumefaciens. The highest cytotoxicity was recorded for EtOAc and Me2CO extracts with the lowest relative and absolute IC50 values between 88 and 102 µg/mL, while EtOH extract was the least toxic with predicted relative IC50 value of 1578 µg/mL. Our results indicate that all tested extracts at concentration considered as non-toxic can be one of great importance in combat towards phytopathogenic and human pathogenic strains, as well as natural sources of antimicrobials.

Linden tea has been used in Serbian folk medicine for centuries to induce sweating for colds, relieve throat irritation and cough, reduce blood pressure, as well as a diuretic, spasmolytic, and as a sedative. The main aim of this study was to determine the comprehensive metabolite profile, free radical scavenging activity, and antimicrobial activities against 23 human and plant pathogens of both commercial and field-collected linden tea samples from Serbia. Developed high-performance thin-layer chromatography (HPTLC) method allowed identification of eight major metabolites in investigated samples, while forty-six metabolites were tentatively identified using high resolution mass spectrometry (HRMS): 14 phenolic acids and their derivatives, 15 glycosides, 9 flavonoids, and 8 procyanidins. Both commercial and field-collected linden tea samples showed similar total phenolic content and radical scavenging activity. Seven compounds such as p-hydroxybenzoic acid 4-O-hexoside, caffeic acid cinnamyl ester, pinocembrin, galangin, luteolin 7-O-glucuronide (and its isomer), isorhamnetin hexosyl hexoside were found in Tilia samples for the first time. Radical reagent 2,2-Diphenyl-1-picrylhydrazyl· (DPPH·) was used in DPPH-HPTLC assay which identified chlorogenic acid, caffeic acid, astragalin, quercitrin, tiliroside, and rutin as compounds that exhibit radical scavenging activity. Tilia samples showed inhibitory effect overwhelming only on Gram-positive bacteria, especially on Bacillus subtilis with the lowest MIC values observed, as well as towards Staphylococcus aureus and oral cavity isolates of Streptococcus pyogenes and Streptococcus mutans. Vaginal isolate of Candida glabrata showed more susceptibility than Candida albicans isolate. Generally, extract from Tilia cordata Miller (L4) showed the highest antimicrobial activity against the most of the tested pathogens, among all field-collected Tilia samples.