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Predmet ove doktorske disertacije je proučavanje sekundarnih metabolita odabranih biljnih vrsta roda Euphorbia koje nastanjuju naše prostore. Za ispitivanje su odabrane Euphorbia palustris L. (barska mlečika), Eupohorbia lucida Waldst. & Kit. (visoka mlečika) i Euphorbia nicaeensis All (uskolisna mlečika). Biljne vrste roda Euphorbia poznate su od davnina i našle su svoju praktičnu primenu u tradicionalnoj i savremenoj medicini. Za sve predstavnike ovog roda karakteristično je postojanje biljnog soka (lateks) koji se skadišti u posebnim ćelijama koje se nalaze u stablu i listovima biljaka. Lateks predstavlja vodenu emulziju smeše terpena, fenolnih jedinjenja, cerebrozida, glicerola i drugih jedinjenja koja ima zaštitno dejstvo za biljku. Najzastupljenija jedinjenja u lateksu pripadaju klasi terpena i to diterpenima i triterpenima..., The subject of this doctoral dissertation is the study of secondary metabolites of selected plant species of the genus Euphorbia that grow in our region. Euphorbia palustris L. (swamp spurge), Euphorbia lucida Waldst. & Kit. (shining spurge) and Euphorbia nicaeensis All. (narrow-leaved spurge) were selected for the research. Plant species of the genus Euphorbia have been known since ancient times and have found practical applications in traditional and modern medicine. All the plants of this genus are characterized by the existence of a milky sap (latex) which is stored in specialized cells found in the stem and leaves of the plants. Latex is an aqueous emulsion of a mixture of terpenes, phenolic compounds, cerebrosides, glycerols and other compounds that has a protective effect on the plant. Terpenes are the major components of the latex, namely diterpenes and triterpenes...

Šljiva (Prunus domestica L.) ima izuzetan značaj kao voćarska kultura u Srbiji i tradicionalno se prerađuje u rakiju šljivovicu. S obzirom da je godišnji prinos šljive ugrožen različitim bolestima, naročito izazivačima bakterioznog raka i mrke truleži neophodno je naći efikasne agense za njihovo suzbijanje, a naročito alternativu upotrebi hemijskih pesticida. U ovom radu je prvi put analiziran mikrobiom filosfere četiri sorte šljive (Čačanska lepotica, Čačanska rodna, Požegača i Ranka), obuhvatajući ranu (maj) i kasnu fenološku fazu (juli) razvića ploda. Indeksi alfa i beta diverziteta su pokazali da su zajednice bile heterogene i da je njihov sastav zavisio od sezone uzorkovanja i sorti šljive. U ukupnoj bakterijskoj zajednici je metagenomskim pristupom utvrđeno da su predstavnici razdela Proteobacteria bili najdominantniji, a najzastupljenije vrste su bile iz rodova: Methylobacterium, Sphingomonas i Hymenobacter. Tradicionalnim kultivabilnim pristupom i analizom sekvenci gena za 16S rRNK identifikovane su Pseudomonas syringae i Pseudomonas graminis kao najdominantnije vrste. Analiza mikobioma je ukazala na dominatnu zastupljenost predstavnika razdela Ascomycota. Vrste iz rodova Aureobasidium i Cryptococcus su bile najučestalije u maju, dok su u julu predstavnici različitih rodova dominirali na sortama šljive (Cryptococcus, Metschnikowia, Fusarium i Hanseniaspora). U okviru kolekcije kultivabilnih gljiva, identifikovano je 11 izolata Monilinia laxa na osnovu filogenetske analize sekvenci ITS regiona. Odabrani izolati M. laxa su prouzrokovali tipične simptome na ubranim plodovima sorti Požegača i Čačanska rodna, a testiranjem potencijalnih bakterijskih patogena su izdvojeni izolati P. syringae ČL2/2 i P7/16_2, koji su izazivali nekroze na ubranim listovima šljive svih ispitivanih sorti. Analizom antagonističkog potencijala kolekcije bakterija i kvasaca poreklom sa sve četiri sorte je izdvojen izolat Bacillus thuringiensis R3/3 sa snažnom antibakterijskom aktivnošću, Plum (Prunus domestica L.) has a great importance as a fruit crop in Serbia and it is traditionally processed into slivovitz brandy. Considering that the annual plum yield is significantly affected by various pathogens, especially those causing bacterial canker and brown rot, it is necessary to find effective agents for their control, and in particular, an alternative to the chemical pesticides. In this research, microbiome of four plum cultivars phyllosphere (Čačanska lepotica, Čačanska rodna, Požegača i Ranka) was analyzed for the first time, including the early (May) and late phenological stages (July) of fruit development. Alpha and beta diversity indices had shown that the communities were heterogenous and that their composition depended on sampling season and plum cultivars. The metagenomic approach revealed that the representatives of the phylum Proteobacteria were the most dominant in the total bacterial community, and that the most represented species were from the genera: Methylobacterium, Sphingomonas and Hymenobacter. Traditional cultivable approach and 16S rRNA sequence analyses identified Pseudomonas syringae i Pseudomonas graminis as two most dominant species. Mycobiome analysis revealed the dominance of the phylum Ascomycota representatives. Aureobasidium and Cryptococcus species were the most abundant in May, while in July representatives of different genera dominated the plum cultivars (Cryptococcus, Metschnikowia, Fusarium and Hanseniaspora). From the collection of cultivable fungi, eleven Monilinia laxa isolates were identified based on phylogenetic analysis of the ITS region sequences. The selected M. laxa isolates caused typical symptoms on the harvested fruits of the Požegača and Čačanska rodna cultivars, and the testing of potential bacterial pathogens revealed P. syringae ČL2/2 and P7/16_2 isolates, which caused necrosis on the harvested plum leaves of all tested cultivars. Antagonistic potential analysis of bacterial and yeast isolates from

Ispitivanje pigidijalnog žlezdanog kompleksa nalazi značajno mesto u karabidologiji, prevashodno sa stanovišta morfologije i taksonomije, ali u novije vreme i ostalih aspekata koji se mogu odnositi na potencijalnu upotrebu primarnih produkata ovih žlezda u biomedicinskim istraživanjima. Ovaj kompleks predstavlja sistem egzokrinih žlezda koji se sastoji od sekretornih lobusa izgrađenih od sekretornih ćelija, gde se sekreti sintetišu, zatim radijalnih sabirnih kanala, glavnog sabirnog kanala, rezervoara, gde se sekreti skladište, i eferentnog kanala, čija je uloga transport sekreta iz rezervoara u spoljašnju sredinu. Primarna funkcija sekreta pigidijalnih žlezda jeste odbrana trčuljaka od potencijalnih predatora iz prirode. U hemijski sastav ovih sekreta ulazi čitav niz jedinjenja koja se uglavnom ubrajaju u aldehide, ketone i organske kiseline. U poslednje vreme postoji interesovanje za istraživanje antimikrobnog i antitumorskog potencijala navedenih smeša sekreta, s obzirom da je prethodno dokazan antimikrobni i antitumorski potencijal pojedinačnih jedinjenja koja se nalaze u njihovom sastavu. Ciljevi ove doktorske disertacije su usmereni ka ispitivanju antimikrobnog i antitumorskog potencijala sekreta pigidijalnih žlezda odabranih vrsta trčuljaka, što može imati značaj u bazičnim biomedicinskim istraživanjima, kao i ka pronalaženju pozadine ovakvih bioloških aktivnosti u ekološkoj funkciji kod ovih insekata, koja bi podobnije bila približena detaljnijom opservacijom pigidijalnih žlezdanih struktura sa morfo-anatomskog aspekta, primenom elektronske i nelinearne mikroskopije, što bi doprinelo i boljem razumevanju fiziologije ovog sistema. U prvom delu doktorske disertacije koji obuhvata biomedicinski aspekt, posmatrana je po prvi put antimikrobna aktivnost sekreta pigidijalnih žlezda 6 odabranih vrsta trčuljaka na humane patogene (osam laboratorijskih i kliničkih bakterijskih sojeva i osam patogenih mikromiceta), kao i citotoksičnost sekreta 4 odabrane vrste trčuljak

Najštetniji patogeni šampinjona čija pojava u gajilištima izaziva velike gubitke u prinosu su gljive Trichoderma spp., prouzrokovači zelene plesni i Lecanicillium fungicola, prouzrokovač suve truleži šampinjona..., The most devastating fungal pathogens of the white button mushroom that induced the greatest yield losses are Trichoderma spp. and Lecanicillium fungicola, causal agents of green mould and dry bubble disease...

European plum (Prunus domestica L.) is a significant commercial crop in Serbia in terms of total fruit production, and is traditionally processed into slivovitz brandy. The brown rot disease caused by Monilinia laxa drastically reduces plum yield almost every year. Fungal communities associated with leaves and fruits of four local Serbian plum cultivars (Požegača, Ranka, Čačanska Lepotica and Čačanska Rodna) were investigated in two phenological stages during early (May) and late (July) fruit maturation. Alpha diversity indices showed that fungal communities were heterogeneous and Beta diversity indicated that autochthonous fungal communities depended upon seasonal changes and the cultivars themselves. The phylum Ascomycota was the most abundant in all samples, with relative abundance (RA) between 46% in the Požegača cultivar (May) and 89% in the Lepotica cultivar (July). The most abundant genus for all plum cultivars in May was Aureobasidium, with RA from 19.27 to 33.69%, followed by Cryptococcus, with 4.8 to 48.80%. In July, besides Cryptococcus, different genera (Metschnikowia, Fusarium, and Hanseniaspora) were dominant on particular cultivars. Among all cultivable fungi, molecular identification of eleven M. laxa isolates from four plum cultivars was performed simultaneously. Bacterial isolates from the plum phyllosphere were tested for their potential antifungal activity against indigenous M. laxa isolates. The most potent antagonist P4/16_1, which significantly reduced mycelial growth of M. laxa, was identified as Pseudomonas synxantha. Further characterization of P4/16_1 revealed the production of volatile organic compounds and phenazine-1-carboxylic acid (PCA). Crude benzene extract of PCA exhibited 57–63% mycelial growth inhibition of M. laxa. LC/MS analysis of the crude extract confirmed the presence of phenazine derivatives amongst other compounds. Scanning electron microscopy revealed morpho-physiological changes in the hyphae of M. laxa isolates caused

Bacterial leaf spot caused by the plant pathogenic bacterium Pseudomonas syringae pv. coriandricola (Psc) was observed on carrot, parsnip, and parsley grown on a vegetable farm in the Vojvodina Province of Serbia. Nonfluorescent bacterial colonies were isolated from diseased leaves and characterized using different molecular techniques. Repetitive element PCR fingerprinting with five oligonucleotide primers (BOX, ERIC, GTG5, REP, and SERE) and the randomly amplified polymorphic DNA-PCR with the M13 primer revealed identical fingerprint patterns for all tested strains. Multilocus sequence analysis of four housekeeping genes (gapA, gltA, gyrB, and rpoD) showed a high degree (99.8 to 100%) of homology with sequences of Psc strains deposited in the Plant-Associated Microbes Database and NCBI database. The tested strains caused bacterial leaf spot symptoms on all three host plants. Host-strain specificity was not found in cross-pathogenicity tests, but the plant response (peroxidase induction and chlorophyll bleaching) was more pronounced in carrot and parsley than in parsnip.

Aim Assessment of biological control of Pseudomonas syringae pv. aptata using crude lipopeptide extracts (CLEs) of two Bacillus amyloliquefaciens strains (SS-12.6 and SS-38.4) and one Bacillus pumilus strain (SS-10.7). Methods and Results The minimum inhibitory concentration (MIC) of CLEs and their combinations against the pathogen and potential interaction between the extracts were determined in vitro. The most effective antibacterial activity was achieved with the CLE from B. amyloliquefaciens SS-12.6, with an MIC value of 0 center dot 63 mg ml(-1). Interactions between CLE combinations were mostly indifferent. The biocontrol potential of CLEs, mixtures of CLEs, and cell culture of B. amyloliquefaciens SS-12.6 was tested on sugar beet plants inoculated with P. syringae pv. aptata P53. The best result in inhibiting the appearance of tissue necrosis (up to 92%) was achieved with B. amyloliquefaciens SS-12.6 cell culture. Conclusion This work demonstrated significant biocontrol potential of the CLE and cell culture of B. amyloliquefaciens SS-12.6 which successfully suppress leaf spot disease severity on sugar beet plants. Significance and Impact of the Study The findings of biocontrol of sugar beet emerging pathogen will contribute to growers in terms of alternative disease control management. This study represents first assessment of biological control of P. syringae pv. aptata.

Background: Oilseed rape (Brassica napus) is an oil-producing crop, significant for vegetable oil and biodiesel production. The phytopathogenic bacterium Xanthomonas campestris pv. campestris (Xcc) causing bacterial leaf blight was recorded for a first time in 2010 on self-growing winter oilseed rape in Serbia. Ever since, it continued appearing on cultivated crops too. Objectives: The aim of this research was to reveal genetic heterogeneity among 25 Xcc isolates, originated from different cultivars of winter oilseed rape obtained in nine-year period (2010-2018) from different localities in Vojvodina region (Serbia) using repetitive sequence-based PCR (rep-PCR) fingerprinting. Methods: Genomic DNA was extracted using CTAB method. Specific primer pair XCF/XCR, amplifying a DNA fragment of hrpF gene from X. campestris was used for rapid preliminary identification of all tested isolates. Rep-PCR using BOX, ERIC and REP-PCR primers was used to detect presence of genetic heterogeneity among Xcc isolates. Results: All 25 tested isolates were preliminary identified as Xcc based on a positive PCR reaction after amplification of 525 bp region using XCF/XCR primers. BOX and ERIC-PCR generated seventeen different DNA fingerprinting profiles, unlike REP-PCR where only nine different profiles were detected. These results show presence of genetic polymorphism among Xcc isolates from winter oilseed rape, which could indicate either their affiliation to some of the identified Xcc races, or formation of a new Xcc ecotype.

Background: A wide variety of different saprophytic and pathogenic bactera, both living epiphytic or endophytic, colonize physically and nutritionally different microhabitats on plants phyllosphere. Xanthomonas campestris pv. campestris (Xcc) is phytopathogenic bacteria causing bacterial blight symptom on oilseed rape (Brassica napus) and black rot on other cultivated Brassica oleaceae vegetables. Objectives: Metagenomic approach was used to reveal differences in microbial communities colonizing the phyllosphere of four different winter oilseed rape varieties with expressed bacterial blight symptoms and one healthy variety, all collected from five different localities in Vojvodina (Serbia) in 2018. Methods: Next generation sequencing (NGS) Illumina MiSeq platform was used to obtain 16S rRNA sequences from five tested samples, and to gain insight into their alpha and beta diversity. Comparative taxonomic analysis of the obtained sequencing metadata was processed bioinformaticaly. Results: Different percetages of bacterial species belonging to three bacterial phyla-Proteobacteria, Firmicutes, and Actinobacteria were dominant in phyllosphere of all five oilseed rape varietes. Bacteria from the phylum Proteobacteria (66-91%) were dominant in diseased varieties, unlike healthy variety where only 38% belonged to this phylum. Genus Xanthomonas (40-74%) was the most abundant in diseased plants phyllosphere. Only 0.2% of Xanthomonas representatives were present in healthy plants, where order Enterobacteriales (Pantoea, Buchnera, and ud Enterobacteriaceae) were the most abundant. The genus Exiguobacterium was the most abundant in healthy variety (47%). Bacteria from phylum Acintinobacteria were least represented, with less then 1% in diseased and up to 12% (ud-Micrococcaceae, Kocuria, Arthrobacter, and ud-Microbacteriaceae) in healthy oilseed rape variety.

Background: Pectolytic Dickeya and Pectobacterium species that cause blackleg, aerial stem rot and soft rot are commercially important pathogens in potato production. Blackleg symptom caused by these bacteria has been recorded in potato production field in Bačka region (Vojvodina, Serbia) in 2018. Objectives: Metagenomic approach was used to reveal microbial communities colonizing the diseased potato tubers with the aim of better understanding of pathogens distribution. Methods: Single colonies of potential pathogenic bacteria were isolated on nutrient agar and checked for pectolytic activity on potato slices. Ten selected bacterial isolates were identified to the species level after PCR amplification with gapA and mdh primers. The 16S rRNA sequences from diseased potato tubers samples were obtained using Next generation sequencing (NGS) Illumina platform. The obtained sequencing data was processed bioinformaticaly. Results: Mixed infection of the diseased potato plants with Dickeya dianthocola and Pectobacterium carotovorum pv. brasiliensis was revealed after pathogen identification with gapA and mdh genes. All isolates caused soft rot symptoms on potato slices. Metagenomic analysis revealed that present bacteria belong to phylum Bacteroidetes (Bacteroides, Empedobacter, Dysgonomonas, Myroides, and ud-Porphyromonadaceae), Firmicutes (Vagococcus, Lactococcus, Clostridium XIVa, Enterococcus, and ud-Acidaminococcaceae) and Proteobacteria (Acinetobacter, ud-Pseudomonadaceae, ud Enterobacteriaceae, Wohlfahrtiimonas, ud-Neisseriaceae, Enterobacter, Providencia, Arcobacter, and Kerstersia) as the most common representives. Genus Pectobacterium was present in 0.5%.

The present study provides insight into the diversity of 147 Xanthomonas campestris pv. campestris (Xcc) isolates obtained from six Brassica oleracea vegetable crops (broccoli, cabbage, cauliflower, collard greens, kale, kohlrabi) and the winter oilseed rape crop Brassica napus, collected from different regions in Serbia in 2014. The XCF/XCR pathovar-specific primer set was used for fast preliminary identification. In repetitive sequence-based PCR (BOX, ERIC and REP) of all isolates, a higher level of genetic diversity was found in winter oilseed rape isolates compared to isolates from the other hosts. ERIC and REP-PCR showed the highest heterogeneity, with 10 and nine banding patterns, respectively. The REP-PCR results showed the highest correlation (70%) with those obtained with multilocus sequence analysis (MLSA), performed with 10 housekeeping genes (fusA, gap-1, gltA, gyrB1, lacF, lepA, rpoD, dnaK, fyuA and gyrB2). Three distinct phylogenetic groups of winter oilseed rape isolates were detected using MLSA. Two genes, gltA and rpoD, showed the greatest ability to identify and discriminate winter oilseed rape Xcc isolates from isolates of the other six hosts. The lepA gene exhibited specific three-nucleotide changes in sequences of some of the isolates. Results of virulence testing of 18 representative isolates showed statistically significant host-pathogen specialization for Xcc isolates from winter oilseed rape, cauliflower, kale and kohlrabi. In conclusion, oilseed rape isolates are more genetically diverse and show greater specialization to their host in comparison to the rest of the tested isolates from other brassica hosts.

The "Pirot 'ironed' sausage" (Pis) is a traditional, dry, fermented product originating from the town of Pirot, situated in southeastern Serbia. This product is made from different types of meat (goat, sheep, beef and donkey) and spices, without additives or starter cultures. Pis is an organic, unprocessed product, without heat or smoke treatment. The aim of this study was to characterize 120 isolates of lactic acid bacteria (LAB) from Pis produced by six different brands during a two-year period using phenotypic and genetic identification. Preliminary characterization of the LAB was based on general morphology and biochemical tests. Repetitive elements such as REP, BOX and GTG(5), found in the genome of these bacteria, and randomly amplified polymorphic DNA-polymerase chain reaction (RAPD PCR) sequences were used for determination of genetic polymorphism. Identification by 16S rRNA gene sequencing showed the presence of only two LAB species, Lactobacillus sakei (76%) and Leuconostoc mesenteroides (24%). However, genetic polymorphism was detected using fingerprinting methods. In comparison with other primers, the profiles obtained with GTG(5) showed the highest heterogeneity for most of the tested isolates, with sequencing results additionally confirming its discriminatory power. In addition, M13 RAPD primer also produced satisfactory separation of the tested isolates.

"Pirot ironed" is a traditional Serbian dry-fermented sausage manufactured in the southeast of Serbia. The changes in the chemical attributes of Pirot ironed sausage were followed during ripening. Samples were taken on the processing days 0, 7, 14, 21 and 28. Pirot ironed sausage was produced from the most valuable cuts of beef and chevon, without addition of starter cultures or fat tissues. Sausages were manufactured in a traditional drying/ripening chamber, where they were pressed every two days to acquire the typical flat form and to speed up the drying. The final water activity was 0.839. The lowest pH value recorded was 5.30 on the processing day 28. During ripening, the water content decreased significantly from 74.72% to 40.32%, while the protein and the fat amounts increased significantly from 19.12% to 45.79% and from 1.22% to 6.21%, respectively. Up to now, the properties of Pirot ironed sausage have not been recognized or published in scientific literature in spite of the long tradition and popularity of this meat product in Serbia.

Ethnopharmacological relevance: Essential oils obtained from resins of Boswellia carteri Birdw. and Commiphora myrrha (Nees) Engl., commonly known as frankincense and true myrrh respectively, have been used extensively since 2800 BCE for the treatment of skin sores, wounds, teeth, inflammation, and urinary tract diseases in traditional medicine; for preparation of mummification balms and unguents; and also as incense and perfumes. Since ancient times, burning of frankincense and myrrh in places of worship for spiritual purposes and contemplation (a ubiquitous practice across various religions) had hygienic functions, to refine the smell and reduce contagion by purifying the indoor air. Aim of the study: The general purpose of the study was to assess the in vitro antimicrobial potential of the liquid and vapour phases of B. carteri and C. myrrha essential oils and burn incense, as well as to test the effectiveness of their in situ application to cleanse microbially-contaminated air within the ambient of an investigated 17th century church. Materials and methods: The chemical composition of B. carteri and C. myrrha essential oils, obtained by hydro distillation of frankincense and true myrrh oleo gum resins was determined using GC/MS, and antimicrobial properties of their liquid and vapour phases were assessed by the broth microdilution and microatmosphere diffusion methods. Chemical analysis of burn incense fume obtained using bottle gas washing with dichloromethane as a solvent was performed by GC/MS, while its antimicrobial activity was evaluated using a modified microatmosphere diffusion method to evaluate germination inhibition for fungi and CFU count reduction for bacteria. The in situ antimicrobial activity of B. carteri burn incense and essential oil vapour phase was assessed in the sealed nave and diaconicon of the church, respectively. Results: The dominant compounds of B. carteri EO were a-pinene (38.41%) and myrcene (15.21%), while C. myrrha EO was charact

Comprehensive analysis of phenolic profiles of botanically different subtypes of Turkish propolis samples were performed using UHPLC-LTQ/Orbitrap/MS/MS method, and additionally total phenolic (TPC) and total flavonoid contents (TFC) as well as their antioxidative activities were evaluated by spectrophotometry. Antimicrobial activity of Turkish propolis against oral cavity bacteria from the genus Streptococcus (S. pyogenes, S. sanguinis, S. mutans) and Candida albicans ATCC 10231 was determined by diffusion and microdilution methods. Extensive fingerprint analysis of Turkish propolis revealed the presence of fifty one phenolic compounds, with fifteen quantified which confirm their affiliation to the two subtypes of the European propolis. All analysed samples have shown antimicrobial potential against all tested bacteria, with S. pyogenes being the most sensitive one. Turkish propolis, especially its orange subtype, can be considered as the high-quality product due to its rich phenolic and flavonoid content, strong antioxidative and antimicrobial activities. Turkish propolis could be, therefore, a good raw material for food and pharmaceutical industry.

Pseudomonas syringae pv. aptata is the causal agent of bacterial leaf spot disease of sugar beet (Beta vulgaris). During 2013, 250 samples were collected from leaf lesions with typical symptoms of bacterial leaf spot in commercial fields of sugar beet in Serbia, and 104 isolates of P.syringae pv. aptata were obtained. Identification and characterization was performed using biochemical, molecular and pathogenicity tests. Identification included LOPAT tests and positive reactions using primers Papt2F and Papt1R specific for P.syringae pv. aptata. Repetitive (rep) sequence-based PCR typing with ERIC, REP and BOX primers revealed high genetic variability among isolates and distinguished 25 groups of different fingerprinting profiles. Pulse-field gel electrophoresis (PFGE) and multilocus sequence analysis (MLSA) of representative isolates showed higher genetic variability than in rep-PCR analysis and distinguished three and four major genetic clusters, respectively. A pathogenicity test performed with 25 representative isolates on four cultivars of sugar beet confirmed the occurrence of leaf spot disease and showed correlation between the most aggressive isolates and the genetic clusters obtained in MLSA. All these findings point to the existence of several lines of P.syringae pv. aptata infection in Serbia that are genetically and pathologically different.

In the light of growing interest in discovering new sources of natural antimicrobial agents, we extracted and tested the efficiency of julid defensive secretions in comparison with commercially used antibiotic and antimycotics. This work involved chemical characterization of the defensive secretions of Cylindroiulus boleti (C. L. Koch, 1847), Megaphyllum bosniense (Verhoeff, 1897) and M.unilineatum (C. L. Koch, 1838), as well as in vitro evaluation of their antimicrobial activity against 11 bacteria, one yeast and eight filamentous fungi. Compounds of the analysed defensive secretions included p-benzoquinones, alkyl esters of fatty acids and ketones. Ketones were recorded for the first time in the order Julida, and they were detected in secretions of both Megaphyllum species. All three analysed defensive secretions showed antibacterial and antifungal potential against all of the tested pathogens. Staphylococcus aureus proved to be the most sensitive bacterial strain to all analysed secretions. Also, defensive secretion of M.unilineatum showed significant antibacterial potential against Bacillus subtilis. The most resistant bacterial strains in this study were Escherichia coli, Erwinia persicina and Pseudomonas syringae pv. tomato. Analysed defensive secretions achieved the strongest antifungal activity against Aspergillus parasiticus (secretions of all three millipede species), Penicillium griseofulvum (secretions of C.boleti and M.bosniense) and Cladosporium oxysporum (secretion of M.unilineatum). The most resistant mycromycetes were A.niger (to all tested defensive secretions), A.flavus (to secretion of M.unilineatum) and P.lanosum (to secretions of C.boleti and M.bosniense). Our results showed a generally lower level of activity compared to antibiotic and a significantly higher level compared to antimycotics. The results of this study elucidate and open opportunities for further research in the field of millipede chemical ecology.

Proposed distributed reactivity model of dehydration for seedling parts of two various maize hybrids (ZP434, ZP704) was established. Dehydration stresses were induced thermally, which is also accompanied by response of hybrids to heat stress. It was found that an increased value of activation energy counterparts within radicle dehydration of ZP434, with a high concentration of 24-epibrassinolide (24-EBL) at elevated operating temperatures, probably causes activation of diffusion mechanisms in cutin network and may increases likelihood of formation of free volumes, large enough to accommodate diffusing molecule. Many small random effects were detected and can be correlated with micro-disturbing in a space filled with water caused by thermal gradients, increasing capillary phenomena, and which can induce thermo-capillary migration. The influence of seedling content of various sugars and minerals on dehydration was also examined. Estimated distributed reactivity models indicate a dependence of reactivity on structural arrangements, due to present interactions between water molecules and chemical species within the plant.

In this study the efficacy of two different methods for extracting lipopeptides produced by five Bacillus strains-ethyl acetate extraction, and acid precipitation followed by methanol extraction-was investigated using mass spectrometry. High performance thin layer chromatography (HPTLC) was also used for the simultaneous separation of complex mixtures of lipopeptide extracts and for the determination of antimicrobial activity of their components. The mass spectra clearly showed well-resolved groups of peaks corresponding to different lipopeptide families (kurstakins, iturins, surfactins, and fengycins). The ethyl acetate extracts produced the most favorable results. The extracts of SS-12.6, SS-13.1, and SS-38.4 showed the highest inhibition zones. An iturin analog is responsible for the inhibition of Xanthomonas arboricola and Pseudomonas syringae phytopathogenic strains. HPTLC bioautography effectively identified the active compounds from a mixture of lipopeptide extracts, proving in situ its potential for use in direct detection and determination of antimicrobials. In the test of potential synergism among individual extracts used in different mixtures, stronger antimicrobial effects were not observed. Biochemical and phylogenetic analysis clustered isolates SS-12.6, SS-13.1, SS-27.2, and SS-38.4 together with Bacillus amyloliquefaciens, while SS-10.7 was more closely related to Bacillus pumilus.

The Pirotska “ironed” sausage is a traditional dry fermented product from south-eastern region of Serbia, made of different types of meat (e.g. horse, goat, sheep and beef) and spices, without additives or starter cultures. The flat sausage is a totally organic, unprocessed product without heat and smoke treatment applied. The aim of this study was to characterize 120 isolates of lactic acid bacteria (LAB) from Pirotska sausage produced by six different brands in two-year period using phenotypic and genotypic identification. The phenotypic characterization and preliminary identification of LAB was based on general morphology and biochemical tests (gas production, growth at different temperatures, arginine hydrolysis etc.). The repetitive elements (REP, BOX, (GTG)5-PCR) found in the genome of these bacteria and randomly amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR) using M13 primer were used for determination of genetic polymorphism. Sequencing of 16S rRNA gene was used for species level identification. The 16S rRNA sequencing showed presence of only two species. Lactobacillus sakei was the dominant species (76%), followed by Leuconostoc mesenteroides (24%). However, plenty of genetic polymorphism within these two species was detected using (GTG)5-PCR fingerprinting. The results after comparing fingerprinting patterns of bacterial populations from different brands, due to the use of different meat types as well as different percentages of meat content, revealed some genetic similarity in few clusters and emphasized significant polymorphism within others.

Fitopatogena bakterija Pseudomonas syringae pv. syringae parazitira oko 180 biljnih vrsta, među kojima najveći ekonomski značaj ima na voćnim vrstama, zatim ratarsko-povrtarskim, ali i na nekom ukrasnom bilju. Kod nas je eksperimentalno potvrđena kao parazit kruške, jabuke, kajsije, trešnje, višnje, šljive, maline, breskve, tikve, blitve i graška. Razvojem molekularnih metoda omogućeno je praćenje genetičkog diverziteta populacije ove bakterije poreklom sa različitih domaćina, kao i otkrivanje veza između patogenih varijeteta unutar same vrste P. syringae. Obzirom da je bakterija P. s. pv. syringae izuzetno polifagna i da je utvrđena na velikom broju domaćina kod nas, cilj ovog rada je bio proučavanje populacije ove bakterije poreklom za različitih domaćina analizom sekvenci konzervativnih gena (MLSA). Izolati korišćeni u radu su poreklom sa trešnje (KBNS91 i V-109), višnje (V-85 i V-88), graška (Gr1), blitve (Ps105) i tikve (7/1). Kao referentni korišćen je soj P. s. pv. syringae GSPB 1142 poreklom sa pasulja. Bakterijska suspenzija gustine 107-108 ćel/ml je pripremana u tubicama sa sterilnom vodom u koju su dodavane pojedinačne kolonije izolata gajenih na hranljivom agaru, starosti 48 sati. Zatim je vršena ekstrakcija DNK korišćenjem CTAB metoda. Za analizu sekvence korišćeni su produkti dobijeni amplifikacijom konzervativnih gena gltA, gapA, gyrB i rpoD. Umnožavanje DNK je vršeno prema sledećem protokolu: inicijalna denaturacija 95°C 3 min; denaturacija 94°C 2 min, hibridizacija 56°C 1 min, elongacija 72°C 1 min (35 ciklusa); finalna elongacija 72°C 5 min. Sekvenciranje dobijenih PCR produkata je vršeno u Macrogen Inc. Neighbour-joining filogenetsko stablo je konstruisano korišćenjem Mega7 programa. Filogenetsko stablo konstruisano na bazi sekvenci konzervativnih gena ukazuje na razdvajanje izolata u dva klastera. Jednu grupu čine izolati sa trešnje (V-109) i višnje (V-88), dok drugu grupu čine ostali izolati koji ispoljavaju razlike unutar same grupe. Tako, slič

Recent studies have been made to investigate some molecular features of plant pathogenic bacteria Pseudomonas syringae pv. coriandricola. This pathogen causes bacterial leaf spot disease on some members of family Apiaceae, such as coriander, carrot, parsnip and parsley. Initial symptoms are small water-soaked lesions on foliage, which then develop into spots varying in shades, tan, brown or black. The spots are usually limited by leaf veins having an angular appearance, and visible from both sides of leaves. Under favorable conditions of high humidity, spots may spread and cause foliage blighting. P. s. pv. coriandricola strains used in this study originate from carrot, parsley and parsnip. Repetitive element PCR fingerprinting (rep PCR), using REP, BOX, ERIC, (GTG)5 and SERE primers, and randomly amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR) using M13 primer were used to determinate genetic polymorphism between tested strains. Further molecular features were observed on the basis of a multilocus sequence analysis (MLSA) by sequencing key genes for Pseudomonas syringae (gapA, gltA, gyrB, rpoD). DNA profiling revealed identical banding patterns for all tested strains. The sequence and phylogenetic analysis of three MLSA genes (gapA, gyrB, rpoD) showed homology with pathotype strains P. s. pv. coriandricola deposited in PAMDB (http://genome.ppws.vt.edu/cgi-bin/MLST/home.pl). Sequencing of gltA gene determined the isolates above the species level (P. syringae). Our results indicate that gapA, gyrB and rpoD genes are more discriminatory then gltA and their usage is therefore suggested for further genotypic studies of this pathogen.

Proposed distributed reactivity model of dehydration for seedling parts of two various maize hybrids (ZP434, ZP704) was established. Dehydration stresses were induced thermally, which is also accompanied by response of hybrids to heat stress. It was found that an increased value of activation energy counterparts within radicle dehydration of ZP434, with a high concentration of 24-epibrassinolide (24-EBL) at elevated operating temperatures, probably causes activation of diffusion mechanisms in cutin network and may increases likelihood of formation of free volumes, large enough to accommodate diffusing molecule. Many small random effects were detected and can be correlated with micro-disturbing in a space filled with water caused by thermal gradients, increasing capillary phenomena, and which can induce thermo-capillary migration. The influence of seedling content of various sugars and minerals on dehydration was also examined. Estimated distributed reactivity models indicate a dependence of reactivity on structural arrangements, due to present interactions between water molecules and chemical species within the plant.

Two strains of Bacillus sp., SS-12.6 and SS-13.1, showed very strong antibacterial and antifungal activity against phytopathogens. The PCR analysis showed that both strains have the genes for biosynthesis of iturin, bacillomycin and surfactin. Kinetics of production of antimicrobial substances in these strains showed that synthesis started at the beginning of exponential phase of growth. Maximum of activity was slowly reached at the beginning of stationary growth phase and was maintained until the end of observed period. Ethyl acetate extracts of cell-free supernatants of both strains were particularly active against several postharvest fungal pathogens, in vitro and in vivo, in the experiment with apple fruits. Mass spectrometry analysis of ethyl acetate extract of the supernatant of strain SS-12.6 confirmed the presence of antimicrobial lipopeptide surfactin. (c) 2013 Elsevier Inc. All rights reserved.