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During May 2016, severe blight symptoms were observed in several raspberry and blackberry fields in Serbia. In total, 22 strains were isolated: 16 from symptomatic raspberry shoots, 2 from asymptomatic raspberry leaves, and 4 from symptomatic blackberry shoots. Additionally, eight raspberry strains, isolated earlier from two similar outbreaks, were included in the study. Pathogenicity of the strains was confirmed on detached raspberry and blackberry shoots by reproducing the symptoms of natural infection. The strains were Gram-negative, fluorescent on King’s medium B, ice nucleation positive, and utilized glucose oxidatively. All strains were levan positive, oxidase negative, nonpectolytic, arginine dihydrolase negative, and induced hypersensitivity in tobacco leaves (LOPAT + −−− +, Pseudomonas group Ia). Furthermore, all strains liquefied gelatin and hydrolyzed aesculin but did not show tyrosinase activity or utilize tartrate (GATTa + + − −). Tentative identification using morphology, LOPAT, GATTa, and ice-nucleating ability tests suggested that isolated strains belong to Pseudomonas syringae. The syrB gene associated with syringomycin production was detected in all strains. DNA fingerprints with REP, ERIC, and BOX primers generated identical profiles for 29 strains, except for strain KBI 222, which showed a unique genomic fingerprint. In all, 9 of 10 selected strains exhibited identical sequences of four housekeeping genes: gyrB, rpoD, gapA, and gltA. Five nucleotide polymorphisms were found in strain KBI 222 at the rpoD gene locus only. In the phylogenetic tree based on a concatenated sequence of all four housekeeping genes, strains clustered within phylogroup 2 (i.e., genomospecies 1) of the P. syringae species complex, with pathotype strains of P. syringae pv. aceris and P. syringae pv. solidagae as their closest relatives. There was no correlation between genotype and geographic origin, particular outbreak, host, or cultivar.

Acidovorax citrulli, the causal agent of bacterial fruit blotch (BFB), is an economically important pathogen of watermelon and related plant species worldwide. In the period 2014–2018, several outbreaks of BFB were observed in major watermelon production regions in Serbia. A total of 43 strains, isolated from symptomatic watermelon tissue, were analyzed by biochemical, pathogenic and molecular tests. Based on the phenotypic characteristics, PCR assay, and 16S rRNA gene sequence analysis, all strains were identified as A. citrulli. A multilocus sequence analysis of the four housekeeping (adk, gyrB, pilT and gltA) and three virulence genes (Aave_1548, avrRx01 and luxR) revealed that studied A. citrulli strains represent the homogeneous population and they clustered together with group II reference strain AAC00-1. Strain affiliation to group II was confirmed by PCR based on the putative type III secretion effector gene and by duplex PCR test. High homogeneity of studied strains was also confirmed by BOX-PCR. Differences were observed for two strains in their pathogenicity as well as susceptibility to copper compounds. Moreover, six major watermelon varieties grown in Serbia showed high sensitivity to the pathogen, while cross inoculation assay revealed that the strains were able to infect other species within the Cucurbitaceae family.

UVOD. Mučnina, neprijatan osećaj u predelu grla i/ili želuca, koji može i ne mora dovesti do povraćanja, je svesno prepoznavanje podsvesne ekscitacije u oblasti medule, blisko udružene sa delovima centra za povraćanje. Mučnina može biti uzrokovana: iritativnim impulsima iz gastrointestinalnog trakta, impulsima koji potiču iz donjih moždanih struktura, povezanim sa bolešću vožnje i impulsima iz cerebralnog kortkesa. Lekovi izazivaju mučninu u različitoj meri, što je od značaja kada treba izabrati terapiju kod pacijenta koji mučninu teško podnose i skloni su odustajanju od dalje terapije. Apstraktne koncepte (kakav je i mučnina) je teško kvantitativno izmeriti, i u tu svrhu koristimo upitnike. CILjEVI RADA. Studija je imala tri cilja: (1) prevođenje i transkulturološka validacija dela upitnika o gastrointestinalim simptomima iz ugla pacijenta (PROMISGSS deo koji se odnosi na mučninu i povraćanje); (2) razvoj i ispitivanje sopstvenog upitnika za procenu mučnine posle primene lekova, kroz utvrđivanje pouzdanosti, validaciju i faktorsku analizu; (3) utvrđivanje faktora koji doprinose prekidu terapije zbog ekstremne mučnine kod pacijenata na terapiji gvožđem. METODOLOGIJA. Skala koja meri gastrointestinalne simptomime iz ugla pacijenta (PROMIS-GSS deo koji se odnosi na mučninu i povraćanje), prevedena je na srpski jezik i izvršena je transkulturološka validacija iste. Prevod i kulturološka adaptacija skale urađena je u skladu sa preoprukama datim u ISPOR (International Society for Pharmacoeconomics and Outcomes Research) vodiču. Razvijen je i sopstveni upitnik za procenu mučnine posle primene lekova (DINS-Drug Induced Nausea Scale), utvrđeni su njegova pouzdanost, validnost i sprovedena faktorska analiza. Ispitivanje pouzdanosti upitnika obavljeno je kroz određivanje koeficijentaIV Kronbahova alfa za ceo upitnik, Spirman-Braunovog koeficijenta i srednje vrednosti i varijanse za svako pitanje iz upitnika. Punovažnost sadržaja upitnika evaluirala je grupa stručnjaka nezavis, INTRODUCTION. Nausea, an unpleasant feeling in the throat and/or the stomach, which may or may not lead to vomiting, represents conscious awareness of the subconscious excitation in the medulla area, close to the brain’s vomiting center. Nausea can be caused by: irritative impulses in the gastrointestinal tract, impulses originating from the lower brain structures responsible for motion sickness, and impulses originating from the cerebral cortex. Medication can cause varying degrees of nausea. Abstract concepts such as nausea are difficult to measure quantitatively, so in order to do so, we use questionnaires. AIMS. The translation and transcultural validation of the part of the questionnaire dealing with gastrointestinal symptoms from the patient’s perspective (PROMIS-GSS), the part dealing with nausea and vomiting; The development and testing of the medication-induced nausea assessment questionnaire, determining its reliability, validation, and factorial analysis; Determining the factors which contribute to a cessation of treatment due to extreme nausea in patients taking iron supplements. A separate medication-induced nausea assessment questionnaire was developed (DINS-Drug Induced Nausea Scale). This instrument was factorially analyzed and has been determined to be reliable and valid. An interesting phenomenon was observed – medication-induced nausea, which is the most common reasonfor the cessation of otherwise effective treatments. A new nausea assessment questionnaire was developed according to the guidelines established by Robert F. The questionnaire’s reliability was assessed through calculation of: Cronbach’s alpha for the questionnaire as a whole, the Spearman-Brown coefficient and the average values and variance for each individual question found in the questionnaire. The validity of the questionnaire was evaluated by an independent panel consisting of three medical specialists from the Clinical Centre of Kragujevac. Criteria validity was tested accordin

Xanthomonas euvesicatoria phage K Phi 1, a member of Myoviridae family, was isolated from the rhizosphere of pepper plants showing symptoms of bacterial spot. The phage strain expressed antibacterial activity to all X. euvesicatoria strains tested and did not lyse other Xanthomonas spp., nor other less related bacterial species. The genome of K Phi D1 is double-stranded DNA of 46.077 bp including 66 open reading frames and an average GC content of 62.9%, representing the first complete genome sequence published for a phage infecting xanthomonads associated with pepper or tomato. The highest genome similarity was observed between phage K Phi 1 and the Xanthomonas oryzae pv. oryzae specific phage OP2. On the other hand, when compared with other members of the genus Bcep78virus, the genome similarity was lower. Forty-four (67%) predicted K Phi 1 proteins shared homology with Xanthomonas phage OP2, while 20 genes (30%) were unique to K Phi 1. Phage K Phi 1, which is chloroform resistant and stable in different media and in the pH range 5-11, showed a high titer storage ability for at least 2 years at +4 degrees C. Copper-hydroxide and copper-oxychloride reduced phage activity proportionally to the used concentrations and the exposure time. UV light was detrimental to the phage strain, but skim milk plus sucrose formulation extended its survival in vitro. The phages survived for at least 7 days on the surface of pepper leaves in the greenhouse, showing the ability to persist on the plant tissue without the presence of the host bacterium. Results of three repeated experiments showed that foliar applications of the unformulated K Phi 1 phage suspension effectively controlled pepper bacterial spot compared to the standard treatment and the untreated control. The integration of the phage K Phi 1 and copper-hydroxide treatments resulted in an increased efficacy compared to the copper-hydroxide alone.

Timley and reliable detection of the hazelnut pathogen Xanthomonas arboricola pv. corylina (Xac) is essential for the production of good quality disease-free planting material. In order to improve knowledge on diagnostic tools for this quarantine bacterium, the effectiveness of laboratory methods recomended by EPPO and additional modified procedures developed within COST873 was compared. Methods included the classical, biochemical, serological, pathogenicity and molecular approach. To confirm the presence of the pathogen in plant tissue, a combination of different techniques is necessary. For successful isolation of Xac cells from different hazelnut organs, general nutrient agar or semi-selective glucose amended media are recommended. Traditional biochemical techniques are still necessary for the identification of isolates at the species level. ELISA and IF are useful as screening assays for the early and rapid pathogen detection. In addition, PCR-based methods proved to be useful for the rapid detection of Xac cells, although highly pathovar-specific genetic tool is not available. Identification at the pathovar level requires pathogenicity tests on hazelnut plants that enable confirmation of strains' pathogenic nature and reproduction of symptoms. However, a choice of various pathogenicity testing procedures should be made based on testing material available and type and dynamics of symptom development.

Cucurbits production has a long tradition in our country. In some parts of the country, cucumber, watermelon and melon are the most important agricultural crops. High yield and good profit are often compromized by various biotic and abiotic factors. In years with favorable weather conditions cucurbit bacterial diseases can cause serious damage. This paper describes major bacterial diseases of cucurbit plants, such as angular leaf spot caused by Pseudomonas syringae pv. lachrymans, bacterial fruit blotch caused by Acidovorax citrulli, bacterial spot caused by Xanthomonas campestris, bacterial wilt caused by Erwinia tracheiphila and cucurbit yellow wine disease caused by Serratia marcescens, relatively new and invasive disease across the United States. In addition, bacterial leaf spot, caused by Pseudomonas syringae pv. syringae and Pectobacterium carotovorum subsp. carotovorum, inducing bacterial soft rot, are becoming more important in cucurbit production worldwide., Biljke familije Cucurbitaceae se dugi niz godina gaje širom naše zemlje. U pojedinim krajevima, proizvodnja krastavca, lubenice i dinje predstavlja osnovni izvor prihoda za mnoga poljoprivredna domaćinstva. Visok prinos i ekonomsku dobit često ugrožavaju različiti biotski i abiotski faktori. U godinama sa uslovima povoljnim za nastanak infekcije, bakteriozna oboljenja mogu prouzrokovati velike gubitke. U ovom radu predstavljene su najznačajnije bakterioze biljaka familije Cucurbitaceae kao što su uglasta pegavost lišća krastavca, prouzrokovana bakterijom Pseudomonas syringae pv. lachrymans; bakteriozna mrljavost plodova lubenice, čiji je prouzrokovač Acidovorax citrulli; zatim bakteriozna pegavost koju prouzrokuje Xanthomonas cucurbitae; bakteriozna uvelost krastavca prouzrokovana vrstom Erwinia tracheiphila i žutilo vreža, čiji je prouzrokovač Serratia marcescens. Gubicima u proizvodnji doprinose i prouzrokovači bakteriozne pegavosti, Pseudomonas syringae pv. syringae, kao i bakteriozne vlažne truleži, Pectobacterium carotovorum subsp. carotovorum.

Continuous use of copper-based treatments and antibiotics in pear and apple protection programs may result in appearance of copper or antibiotic resistant strains of Erwinia amylovora. In order to determine the potential occurrence of copper or/and antibiotic resistance in Serbia we studied in vitro effect of different concentrations of copper sulfate, streptomycin and kasugamycin on development and growth of 40 E. amylovora strains originating from this region. Filter sterilized solution of the bactericides was added to the NA medium after sterilization to a final concentration of 100 and 200 ppm, respectively, followed by spot inoculation of each strain on the medium. None of the strains developed on NA amended with 100 or 200 ppm of streptomycin or kasugamycin. Our study showed that, despite the potential exposure to the antibiotics, E amylovora strains from Serbia did not develop resistance to streptomycin or kasugamycin. On the other hand, all strains had normal growth on NA amended with 100 ppm of copper sulfate indicating certain level of resistance to copper ions. At 200 ppm, 28 strains formed colonies of normal size and appearance, while 12 strains had smaller colonies with reduced growth. This might be due to the extensive and frequent use of copper-based compounds to control fire blight in Serbia.

During the summer of 2014, watermelon producers in the north of Serbia (Srem district), observed the occurrence of irregularly shaped, water-soaked lesions on mature watermelon fruits indicating possible bacterial nature of the disease. Bacteria were isolated from symptomatic samples and identified as Acidovorax citrulli, previously described as a causal agent of bacterial fruit blotch. In the past few years, this pathogen caused considerable damage to the watermelon industry of the United States where, in some commercial production sectors, the loss of marketable fruit reached 90%. In Serbia, it is considered a quarantine pathogen and was not recorded until 2014., U leto 2014. godine, proizvođači lubenice iz sremskog okruga uočili su pojavu simptoma u vidu pega vodenastog izgleda i nepravilnog oblika na zrelim plodovima lubenice, koji su ukazivali na oboljenje bakteriozne prirode. Izolacijom i identifikacijom patogena, utvrđeno je prisustvo bakterije Acidovorax citrulli, prouzrokovača bakteriozne mrljavosti plodova lubenice. Ova vrsta je u skorije vreme dosta dobila na značaju, posebno zbog gubitaka u proizvodnji koji su u pojedinim oblastima Sjedinjenih Američkih Država (SAD) dostizali i 90% od ukupnog prinosa. U Srbiji A. citrulli ima karantinski status i nalazi se na A1 listi Evropske i mediteranske organizacije za zaštitu bilja (EPPO).

Tumorigenic strains of Agrobacterium spp. are responsible for crown gall disease of numerous plant species. We present here draft genome sequences of nonpathogenic Agrobacterium nepotum strain 39/7(T) (CFBP 7436(T), LMG 26435(T)), isolated from crown gall tumor on Prunus cerasifera, and tumorigenic Agrobacterium sp. strain KFB 330 (CFBP 8308, LMG 28674), isolated from galls on raspberry.

Bakteriozna plamenjača leske, čiji je prouzrokovač fitopatogena bakterija Xanthomonas arboricola pv. corylina (Xac), najznačajnije je bakteriozno oboljenje ove jezgraste voćke u svetu. U našoj zemlji bolest je prvi put uočena 50-ih godina prošlog veka, nakon čega nije bilo daljih proučavanja ovog patogena. U periodu 2008 - 2011. godine, u nekoliko rejona gajenja leske u Srbiji uočena je pojava simptoma pegavosti lišća, nekroze i uvelosti pupoljaka i letorasta, koji su ukazivali na moguću bakterioznu prirodu oboljenja. Simptomatični uzorci obolelih biljaka prikupljeni su sa različitih lokaliteta u našoj zemlji sa ciljem utvrđivanja etiologije oboljenja. Iz zaraženog biljnog tkiva izolovani su Gram i oksidaza negativni, katalazapozitivni aerobni sojevi bakterija žute boje kolnija na HA podlozi. Za dalji rad odabrano je ukupno 47 reprezentativnih sojeva, koji su proučeni primenom standardnih i savremenih metoda. U istraživanja su uključeni i sojevi poreklom iz Poljske, kao i referentni sojevi Xac poreklom iz međunarodnih kolekcija. Detaljno su proučene morfološke, patogene, biohemijsko-fiziološke, serološke i genetičke odlike sojeva, na osnovu kojih su svi proučavani sojevi identifikovani kao Xanthomonas arboricola pv. corylina. Na podlozi od hranljivog agara (HA) formirane su sitne i sjajne kolonije, dok su one na podlozi obogaćenoj glukozom (GYCA) bile krupne, sluzaste i ispupčene. Svi proučavani sojevi hidrolizuju skrob, želatin i eskulin, koriste glukozu, maltozu, saharozu kao izvor ugljenika, ali ne i sorbitol, metabolišu kvinate, razvijaju se pri 35°C i u prisustvu 2% NaCl. Sojevi su prouzrokovali hipersenzitivnu reakciju na listovima duvana i paradajza i mahunama boranije. Patogenost sojeva potvrđena je testom patogenosti na domaćinu i reprodukcijom simptoma prirodne infekcije. Na osnovu Biolog testa utvrđena je raznovrsnost proučavanih sojeva čiji su metabolički profili bili specifični za rod Xanthomonas..., Bacterial blight of hazelnut, caused by Xanthomonas arboricola pv. corylina (Xac), is the most important bacterial disease of this nut crop worldwide. In our country, after the disease was initially described in the 1950s, there were no further data about the research of this pathogen. During the growing season of 2008-2011, symptoms of leaf spots, bud and twig necrosis and dieback, suggesting bacterial infection, were observed on hazelnut plants in several localities in Serbia. In order to study etiology of disease, symptomatic samples of diseased hazelnut plants were collected from different hazelnut-growing regions in the country. Isolations from the diseased tissue resulted in Gram and oxidase negative, catalase-positive, aerobic yellow bacterial colonies. Total of 47 representative strains were selected for further study. Strains isolated from hazelnut in Poland and various reference strains of Xac originating from international collections were also included in research. Based on their morphological, pathogenic, biochemical, physiological, serological and genetic characteristics tested strains were identified as Xanthomonas arboricola pv. corylina. Bacterial colonies formed on NA medium were small and glistening and on glucose amended GYCA medium large, mucous and domed. All strains hydrolyzed starch, gelatin and esculin, used glucose, maltose, sucrose as a carbon source, but not sorbitol, metabolized quinates, developed at 35 °C in the presence of 2% NaCl. The strains induced hypersensitive reaction of tobacco, tomato leaves and bean pods. Pathogenic nature of the strains was confirmed in pathogenicity test on host plants by reproduction of symptoms similar to natural infection. Based on carbon source utilization, metabolic diversity of investigated strains was confirmed. Strain's metabolic profiles were specific for Xanthomonas genus. Serological methods (ELISA i IF test) indicated similarity in serological characteristics among the studied strains, as well

In search for an alternative to chemical control of fire blight, we isolated seven bacteriophages specific to E. amylovora. Three phages were isolated from water, three from symptomless pear leaves and one from apple leaves with characteristic fire blight symptoms. Host-range studies showed that the phages differed in the ability to lyse 40 strains of E. amylovora isolated in Serbia. Based on its lytic activity, phage Phi Ea2 was chosen for further study of biological control potential in pear and apple blossom bioassays. A phage suspension of 10(8) PFU/ml was applied on blossoms either 2 h before, 2 h after or at the same time of inoculation. In three fully replicated bioassay experiments, application of host-specific phages 2 h before inoculation and at the time of inoculation significantly reduced fire blight symptom development compared to untreated control. However, disease reduction was not significant when phages were applied 2 h after inoculation.

In this study, three bacterial DNA extraction procedures were compared prior to real-time PCR. Healthy pear leaves and twigs were crushed in antioxidant maceration buffer and spiked with Erwinia amylovora to final concentrations from 2.1 x 10(6) to 2.1 x 10(1) cells ml(-1). Bacterial DNA was extracted from aliquots of spiked crude extracts using (i) isopropanol, (ii) REDExtract-N-Amp (TM) Plant PCR kit, and (iii) Taylor's modified DNA purification procedure. The ams region of the chromosomal DNA was selected as target for the real-time PCR. In this study, the REDExtract-N-Amp (TM) and Taylor's modified DNA extraction procedure were most successful in removing PCR inhibitors, leading to detection of 2.1x10(2) E. amylovora CFU/ml. At this concentration, pathogen can be efficiently detected in less than 5 h in spite of inhibitors and plant DNA reducing sensitivity of the reaction. These two methods increased amplification efficiency in real-time PCR compared to a simple isopropanol DNA extraction procedure from plant tissues, where the lowest detected concentration was 2.1 x 10(4) CFU/ml. In our research, real-time PCR has proven to be very sensitive method for detection of E. amylovora in plant material. It was 100 times more sensitive compared to other conventional PCR procedures.

In 2010, a serious outbreak of crown gall disease was observed on grapevines (Vitis vinifera L. cv. Cabernet Sauvignon) in several commercial vineyards located in the Vojvodina province, Serbia. Bacteria were isolated from the young tumor tissue on nonselective YMA medium and five representative strains were selected for further identification. Tumorigenic (Ti) plasmid was detected in all strains by PCR using primers designed to amplify the virC pathogenicity gene, producing a 414-bp PCR product. The strains were identified as Agrobacterium vitis using differential physiological and biochemical tests, and a multiplex PCR assay targeting 23S rRNA gene sequences. In the pathogenicity assay, all strains induced characteristic symptoms on inoculated tomato and grapevine plants. They were less virulent on tomato plants in comparison to the reference strains of A. tumefaciens and A. vitis.