Identification and molecular characterization of Chryseobacterium vrystaatense ST1 isolated from oligomineral water of southeast Serbia

The isolation and molecular characterization of bacterial strains isolated from water sources in the Vlasina Mountain in southeast Serbia, confirmed the presence of a new species Chryseobacterium vrystaatense ST1. This Gram- negative species showed an extremely low level of biochemical reactivity in biochemical tests. The gene for 16S rRNA was amplified by PCR using universal primers and sequenced. Comparison of 16S rRNA gene sequence and phenotypic features indicated that the isolate ST belonged to Chryseobacterium vrystaatense. A BLAST search of sequenced 1088 nucleotides of the 16S rRNA gene with all sequences deposited in the NCBI collection showed the highest similarity (98%) with the strain Chryseobacterium vrystaatense sp. nov., designated as strain R-23533. The very high homology of these two strains allowed classification of our strain at the species level, but some differences indicate, and indirectly confirm, that the isolate ST is an authentic representative. On the basis of these results, we could conclude that Chryseobacterium vrystaatense ST was for first time isolated in Serbia, which is particularly important when one bears in mind that there are only three sequences of this species deposited in the NCBI collection

A method for the rapid detection and identification of halo blight pathogen on common bean

A diagnostic method based on nested-PCR, followed by ELISA and conventional bacteriology tests, for the rapid and reliable detection of halo blight pathogen Pseudomonas savastanoi pv. phaseolicola (Psp) collected from infected bean leaves and seeds is described. Psp formed white, small and flat colonies on nutrient agar medium, creamy white, flat and circular on Milk-Tween agar medium and light yellow, convex and shiny on modified sucrose peptone agar medium. Eighteen Gram-negative, catalase-positive and oxidase-negative strains were subjected to nested PCR with primers P 5.1/P 3.1 and P 5.2/P 3.2, which directed the amplification of the 450 bp target DNA fragment in all tested strains. According to the results of DAS-and PTA-ELISA with respect to reactivity to specific antibodies, all analyzed strains belonged to Psp bacterium. Pathogenicity was tested on bean pods and cotyledon leaves, on which greasy spots were formed. Psp did not cause hypersensitive reaction on the leaves of tobacco and geranium. Strains produced levan, fluorescent pigment, oxidative metabolism of glucose, did not reduce nitrate, did not produce indole and H2S, did not hydrolyze starch, gelatin and esculin; they produced acid from glucose, mannose, sucrose and glycerol, and did not produce acid from maltose, starch, esculin, dulcite, sorbitol, inositol and erythritol.This article has been retracted. Link to the retraction: [http://dx.doi.org/10.2298/ABS150609074E

Fenotipska i genetička obilježja osjetljivih i višestruko otpornih izolata Pseudomonas aeruginosa u južnoj Srbiji

Drug resistance of Pseudomonas aeruginosa is a leading problem in hospital infections. The aim of this study was to determine the best molecular genetic discrimination method for Pseudomonas spp. isolates among 94 outpatients and inpatients and see their grouping by phenotype characteristics (biofilm formation, frequency of serotypes, pigmentation, production of different class of beta-lactamases, and susceptibility to different antibiotic classes) and genotype. The most common serotypes were P1, P6, and P11, while co-productions of pyoverdine and pyocyanin were observed in 70 % of isolates. A total of 77.66 % isolates were mostly weak and moderate biofilm producers. Isolates were susceptible to colistin (100 %), aztreonam (97.87 %), imipenem (91.49 %), doripenem (90.43 %), and meropenem (84.04 %). MICs values confirmed susceptibility to ceftazidime and cefepime and singled out meripenem as the most effective inhibitor. Most isolates were resistant to aminoglycosides and fluoroquinolones. Only two isolates produced ESBL, eight were carbapenemase producers, and five isolates produced MBLs. Twenty-nine isolates were multidrug-resistant; 82.8 % of which produced both pigments, 58.3 % were non-typeable, while the P6 and P11 serotypes were equally distributed (16.7 %). Thirteen MDR isolates were strong enzyme producers. RAPD PCR analysis using primer 272 proved the best at discriminatory fingerprinting for Pseudomonas isolates, as it allocated 12 clusters. A correlation between DNA patterns and antibiotic resistance, production of pigments, serotypes distribution, and biofilm formation was not observed, and only confirmed higher genetic heterogeneity among P. aeruginosa isolates, which suggests that other molecular methods are needed to reveal potential relations between genotypic patterns and phenotypic characteristics.Antibiotska rezistencija Pseudomonas aeruginosa vodeći je problem u bolničkim infekcijama. Cilj ovoga istraživanja bio je utvrditi najbolju diskriminatorno molekularno-genetičku metodu među 94 ambulantna i bolnička Pseudomonas spp. izolata kako bi se uvidjelo njihovo grupiranje u smislu različitih fenotipskih obilježja (stvaranje biofilma, učestalost serotipova, pigmentacija, proizvodnja različitih klasa beta-laktamaza i osjetljivost na različite skupine antibiotika) u skladu s genotipom. Najčešći serotipovi bili su P1, P6 i P11, a proizvodnja i pioverdina i piocijanina primijećena je kod 70 % izolata. Ukupno 77,66 % izolata uglavnom je iskazalo slabu i umjerenu proizvodnju biofilma. Izolati su bili osjetljivi na kolistin (100 %), aztreonam (97,87 %), imipenem (91,49 %), doripenem (90,43 %) i meropenem (84,04 %). Vrijednosti MIC-ova potvrdile su podložnost izolata ceftazidimu i cefepimu, a izdvojile su meropenem kao najučinkovitiji inhibitor. Većina izolata bila je otporna na aminoglikozid i fluorokinolon. Samo dva izolata proizvela su ESBL, osam izolata sintetiziralo je karbapenemaze, a pet izolata imalo je sposobnost proizvodnje MBL-a. Dvadeset devet izolata bilo je višestruko rezistentno na antibiotike, od kojih je 82,8 % proizvodilo oba pigmenta, 58,3 % bili su netipabilni, a serotipovi P6 i P11 bili su podjednako zastupljeni među njima (16,7 %). Trinaest MDR izolata bili su snažni proizvođači enzima. RAPD PCR analiza korištenjem 272 početnica pokazala se kao najbolja diskriminatorna metoda otiskom prsta (fingerprinting) za Pseudomonas izolate, izdvajajući čak 12 različitih klastera. U ovom istraživanju nije zabilježena povezanost između DNA obrazaca i otpornosti na antibiotike, proizvodnje pigmenata, distribucije serotipova i stvaranja biofilma, što potvrđuje puno veću genetičku heterogenost unutar samih izolata P. aeruginosa, pod čim se podrazumijeva uključivanje drugih molekularnih metoda u otkrivanju potencijalnih odnosa između genetičkih obrazaca i fenotipskih obilježja

Proučavanje raznovrsnosti bakterije Pseudomonas syringae poreklom sa različitih voćaka u Srbiji

Pseudomonas syringae is a widespread and economically important plant pathogen, one found on a number of hosts, including fruit trees, field crops, vegetables, and ornamental plants. This bacterium has been experimentally identified as a parasite of pear, apple, apricot, peach, cherry, sour cherry, plum, and raspberry. The present study was designed to establish differences between strains isolated from fruit trees in Serbia. The pathogenic and biochemical characteristics of isolates were studied. The BOX-PCR method was used to generate genomic fingerprints of Pseudomonas syringae isolates and to identify strains that were previously not distinguishable by other classification methods. Different Bacillus sp. strains were tested for in vitro inhibitory activity against Pseudononas syringae isolates. Bacillus sp. strains show inhibitory activity only against P. syringae isolates that originated from peach. The obtained results demonstrate that the population of the bacterium Pseudomonas syringae from the fruit trees in Serbia is very diverse.Pseudomonas syringae je široko rasprostranjena i ekonomski značajna fitopatogena bakterija, sa širokim krugom domaćina koji uključuje voćke, ratarske, povrtarske i ukrasne biljke. Pseudomonas syringae u Srbiji je eksperimentalno potvrđen kao parazit kruške, jabuke, kajsije, breskve, trešnje, višnje, šljive i maline. Cilj ove studije bio je da utvrdi postojanje eventualnih razlika između sojeva izolovanih sa različitih vrsta voćaka u Srbiji. Proučavane su patogene i biohemijske osobine sojeva. BOX-PCR je korišćen za dobijanje profila izolata Pseudomonas syringae u cilju identifikacije sojeva koji se ne mogu utvrditi drugim metodama. Različiti sojevi roda Bacillus su testirani u cilju utvrđivanja njihove in vitro inhibitorne aktivnosti. Sojevi roda Bacillus su pokazali inhibitornu aktivnost samo na P. syringae izolovanih sa breskve. Dobijeni rezultati pokazali suda je populacija bakterije Pseudomonas syringae poreklom sa voća u Srbiji vrlo raznovrsna

MONITORING OF POTATO FIELDS TO PRESENCE OF RALSTONIA SOLANACEARUM

BACKGROUNDS: Ralstonia solanacearum race 3 biovar 2 is one of the most important plant pathogenic bacteria because of its persistence, wide host range and widespread. It´s causing bacterial wilt of over 250 plant species, including many cultivated crops (potato, tomato, eggplant, geranium, ginger, banana, etc.) and brown rot of potato. OBJECTIVES: The aim of this study was to determine presence of R. solanacearum based on several years monitoring of potato tubers. METHODS: During four-year period (2013-2016), 98 samples of potato tubers (11 cultivars), from 13 localities in Bačka and 1 in Podunavlje region were examined. Isolation from potato tubers with brown rot symptom was performed on semi-selective SMSA medium. PCR method using Ps-1/Ps-2 and OLY1/Y2 primers and immunofluorescence were used to detect presence of R. solanacearum. Pathogenicity was tested on young tomato plants. RESULTS: In 2013, R. solanacearum was detected in 1 sample of potato tuber (Lady Claire) from Srpski Miletić; in 2015 in 2 (Crisps4all, VR 808) from Srpski Miletić and 1 (VR 808) from Stapar; during 2016 in 2 (Crisps4all, Brooke) from Srpski Miletić, 2 (Lady Claire, VR 808) from Stapar, 2 (Lady Claire, Pirol) from Sombor and 1 (Panda) from Boleč. Milky-white, with pink to red centre, flat, irregular, fluidal colonies formed after 3-4 days of incubation on SMSA medium. A 553 bp and 288 bp fragments of 65 representative isolates were amplified using Ps-1/Ps-2 and OLY1/Y2 primer pairs, respectively. IF test proved presence of fluorescent bacterial cells. All strains caused wilting on tomato seedlings four days after inoculation. CONCLUSIONS: The results of this study detected presence of R. solanacearum on 11 potato samples isolated from four locations during period from 2013-2016

Characterisation of New Bacillus circulans Strain Isolated from Oil Shale

Novi soj vrste Bacillus otkriven je tijekom demineralizacije uljnoga škriljevca radi dobivanja kerogena. Učinak demineralizacije izoliranoga soja bio je kudikamo veći od učinka soja Bacillus circulans Jordan 1890. Prema biokemijskim značajkama, sastavu i strukturi proteina, sastavu masnih kiselina, kao i profilu genoma te sekvencija 16S rDNA novi je soj identificiran kao Bacillus circulans VD01.A new strain of Bacillus sp. was obtained during experiments of oil shale demineralization, which were carried out in order to get ’pure’ organic matter (kerogen). The demineralization efficiency of newly isolated strain was found to be substantially higher in comparison with that of Bacillus circulans Jordan 1890. On the basis of the biochemical characteristics, protein patterns and fatty acid composition, as well as the whole genome profile and 16S rDNA sequencing, the new strain was identified as Bacillus circulans VD01

Genetic characterization of pathogenic fluorescent pseudomonads isolated from necrotic cherry and plum buds in Serbia

During past few years a symptoms of plum and cherry bud necrosis were observed in some regions with significant cherry production in Serbia. Gram negative, fluorescent, oxidative bacterial strains were isolated from the margin of necrotic tissue. All investigated strains are levan and HR positive, while negative results are recorded in oxidase, pectinase and arginin dihydrolase tests (LOPAT+---+). Symptoms similar to those observed in natural infection were obtained after artificial inoculation of cherry leaf scares and dormant one year old cherry shoots. Investigated strains as well as reference strain of P. syringae pv. morsprunorum cause the superficial necrosis on artificially inoculated immature cherry fruits, but negative results were recorded in immature pear and lemon fruit tests as well as syringae leaves and bean pods. Gelatin and aesculin tests were negative and tyrosinase and tartrate were positive. Investigated strains isolated from necrotic cherry buds had identical REP-PCR pattern with reference strain of P. syringae pv. morsprunorum. On the basis of obtained results, it was concluded that this bacterium is causal agent of cherry trees bud necrosis in Serbia. [Projekat Ministarstva nauke Republike Srbije, br. 31018 i br. 173026

Genetic diversity of pseudomonas syringae pv. Syringae isolated from sweet cherry in southern and northern regions in Serbia

Bacterial canker and leaf spot caused by plant pathogenic bacterium Pseudomonas is among the most destructive cherry diseases worldwide. Nowadays in Serbia, sweet cherry production significantly increased and the new plantations, mainly grown from imported planting material are being raised every year. During spring, 2018 and 2019, occurrence of bacterial canker and leaf spot symptoms was observed on a newly planted sweet cherry plantations in two localities, Zitorada (Southern region) and Karavukovo (Northern region-Vojvodina). Typical P. syringae colonies were isolated on Nutrient Sucrose Agar supplemented with 5% sucrose (NSA). A total of fifteen isolates were selected and identified. Results of the LOPAT test (+---+) determined them to belong to fluorescent Pseudomonas Group Ia, while results of G(+)A(+)T(-)Ta(-) tests indicate presence of Pseudomonas syringae pv. syringae. Pathogenicity was confirmed on immature sweet and sour cherry fruitlets by forming of black, sunken lesions for all tested isolates. Genes syrB and syrD were successfully detected in all tested isolates. DNA sequencing using gapA, gltA, gyrB and rpoD housekeeping genes determined tested isolates to belong to P. s. pv. syringae using the National Center for Biotechnology Information (NCBI) nucleotide BLAST. The Serbian isolates shared 99.47% to 100% (Zitorada) and 99.38% to 100% (Karavukovo) identity with bacterium P. s. pv. syringae. Phylogenetic analysis grouped isolates from Zitorada in one tree cluster, separate from the Karavukovo isolates,indicating presence of two genetically diverse groups of causal pathogen P. s. pv. syringae, obtained from two geographically distinct localities in Serbia. Phylogeographic analysis grouped isolates from Zitorada in multilocus haplotype coded as REz and isolates originated from Karavukovo in multilocus haplotype coded as REk. Considering that during last few years P. syringae continuously occurs mainly in young sweet cherry plantations, where imported material is used for raising, health status check is recommended to be included as obligatory measure when nursery material is used from import

Supplementary data for article: Dimkic, I.; Stankovic, S.; Nišavic, M.; Petkovic, M.; Ristivojevic, P.; Fira, D.; Beric, T. The Profile and Antimicrobial Activity of Bacillus Lipopeptide Extracts of Five Potential Biocontrol Strains. Frontiers in Microbiology 2017, 8 (MAY). https://doi.org/10.3389/fmicb.2017.00925

Supplementary material for: [https://doi.org/10.3389/fmicb.2017.00925]Related to published version: [http://cherry.chem.bg.ac.rs/handle/123456789/2463