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Xanthomonas arboricola pv. corylina is the causal agent of bacterial blight of hazelnut. The bacterium has been listed as an A2 quarantine pathogen in Europe since 1978 and on the regulated non-quarantine pest list since 2019. Three isolates from various geographic regions and isolated at different times were sequenced using a hybrid approach with short- and long-read technologies to generate closed genome and plasmid sequences in order to better understand the biology of this pathogen.

Bacterial diseases of apple are less numerous compared to fungal diseases, but under favorable conditions they can cause enormous damage. This is especially true for fire blight of apple, which can, in some years, cause complete tree death of sensitive cultivars. Fire blight has been present in Serbia for more than 30 years. Growing sensitive apple cultivars and lack of effective bactericides caused the disease to spread in a relatively short time in most of the apple fruit producing regions. Additional troublesome event for producers in Serbia is the ban on the use of antibiotics in plant protection. In addition to fire blight, this paper also describes other bacterial diseases of apple, such as crown gall, caused by Agrobacterium tumefaciens and Rhizobium rhizogenes, and bacterial dieback and canker of fruit trees, caused by Pseudomonas syringae pv. syringae. The aim of this paper is to provide the knowledge on the most important bacterial diseases of apple, their importance in apple production, epidemiology and disease symptoms, as well as protection measures in order to reduce the damage they cause.

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.

In Montenegro, stone fruit species are grown on intensive and semi-intensive commercial plantations. However, almond production is mainly organized on family gardens and for household consumption. During two seasons (2017–2018), we surveyed apricot, peach, nectarine, sweet cherry, Japanese plum, and almond orchards for the presence of bacterial diseases at different geographical locations in Montenegro. From leaf, petiole and fruit lesions, branch or twig cankers, and necrotizing buds, a total of 29 isolates were obtained and subjected to identification based on their morphological, pathogenic, biochemical, and molecular characteristics. Pathogenicity of the isolates was confirmed by reproducing the symptoms on leaves, fruits, and twigs of the corresponding host plants. The biochemical tests indicated that the isolates belong to Pseudomonas syringae. However, isolates’ characterization showed variation in their phenotypic and molecular features. The presence of the syrB gene and ice nucleation activity grouped most of the isolates within pathovar syringae. The results of rep-PCR using the BOX primer revealed high genetic diversity of isolates. Multilocus sequence analysis (MLSA), using four housekeeping genes, showed that 27 isolates belong to the genomic species 1, P. syringae sensu stricto, corresponding to P. syringae phylogroup 2. However, isolates from the same phylogroup 2 did not form a monophyletic group. One strain isolated from apricot was most distinct and similar to members of genomic species 2, phylogroup 3. All tested isolates showed significant levels of resistance to copper sulfate and high level of sensitivity to streptomycin sulfate in vitro.

"Candidatus Liberibacter solanacearum" is a gram-negative, fastidious bacterium that inhabits and multiplies in the phloem of host plants. The bacterium causes economically important diseases of solanaceous crops such as "zebra chips" of potatoes, and chlorotic weakening of cultivated plants of the Apiaceae family. Beside potatotes, this pathogen causes significant economic losses in tobacco, peper and tomato production. Insect vectors classified in the family Triozidae play an important role in the epidemiology of this bacterium. Insects transfer bacterial cells from infected to healthy plants by feeding on the plant juice from the phloem tissue. This pathogenic bacterium could be transmitted by infected seeds which result in long-distance spread. Based on the analysis of single nucleotide polymorphism (SNP), nine haplotypes (A, B, C, D, E, F, G, H, U) from different geographical regions, host plants and insect vectors have been described. Considering to impossibility of isolation of this bacterium on a nutrient medium, detection and identification are based on the application of molecular methods (conventional PCR, real-time PCR, multiplex PCR, nested PCR and LAMP PCR). Taking into consideration the increasing spread of "Candidatus Liberibacter solanacearum" in Europe, as well as its recent detection in Serbia, the aim of this paper was to raise an awareness about importance of this bacterium and summarize the protocols and molecular methods for its detection and identification.

Crop yield loss due to bacterial plant pathogens need to be reduced to increase global food production demand. Currently available disease management strategies involving copper-based bactericides and antibiotics are losing efficacy due to development of resistance in bacteria. There is long familiar demand of environmentally friendly and sustainable strategies to control bacterial diseases. Bacteriophages are virus that kill target bacteria without affecting another microorganism and environment. Bacteriophage efficiency on phyllosphere is mainly affected by ultraviolet (UV) light. Use of combination of phage, mixture with phage carrier bacteria and optimizing time of application helps in persistence of bacteriophage. There are several bacteriophage products already available in the market to control destructive bacterial diseases. Unlike chemical based traditional control measure, bacteriophage mixture can be easily amended to reduce resistance development in bacteria. In this chapter, the authors discuss from phage isolation to interaction with bacteria and control mechanism of plant diseases.

Bacterial fruit blotch and seedling blight, caused by

Three PCR methods, referred in this study as „conventional“, „nested“ and „chromosomal“ PCR and suggested for routine detection of Erwinia amylovora in pure culture and plant material, were evaluated according to their specificity and sensitivity. Specificity of PCR methods was analyzed by using 42 strains of E. amylovora, originating from different locations and plant species, with diverse PFGE profiles, representing distant populations of the pathogen. Sensitivity of PCR protocols in pure culture was studied by using nine different concentrations of E. amylovora in sterile ultrapure water as a template in PCR reactions. In order to study inhibitory effect of plant DNA and other inhibitors on sensitivity of the three PCR methods bacterial dilutions were mixed with plant macerate of pear, apple and quince prior to the PCR reaction. In specificity assays, tested PCR protocols were able to detect all E. amylovora strains regardless of the host of the strain, its origin or PFGE group, indicating primer specificity. On the other hand, sensitivity among tested methods varied, depending on bacterial concentration and selected plant material used in the PCR. When working with pure cultures nested PCR showed the greatest sensitivity by detecting 1.9 bacterial cells per PCR reaction, followed by detection limit of 9.5 cells per PCR reaction with conventional PCR and 1.9´105 cells/PCR reaction with chromosomal PCR. In spiked samples plant inhibitors either did not affect or they decreased the sensitivity of the PCR reaction, depending on the protocol and/or type of plant macerate. In our experiments, inhibitors from pear and quince macerates did not affect sensitivity of nested PCR, while apple macerate reduced its sensitivity by a factor of 10. Conventional PCR protocol was able to detect 95 cells/PCR reaction in pear and apple macerate, but only 9.5´103 cells/PCR in quince macerate. Greatest decrease in sensitivity of the PCR method was observed in spiked samples with chromosomal PCR since bacterial DNA was not detected in each of the spiked samples. Our research shows that all three PCR protocols are specific for detection of E. amylovora, but nested PCR proved to be most sensitive when working with pure cultures and plant material. [Project of the Serbian Ministry of Education, Science and Technological Development, Grant no. III46008]

Xylella fastidiosa (Xf) is one of the most important bacterial pathogens worldwide causing several diseases in more than 350 cultivated and wild plant species. The bacterium is listed as a quarantine organism in the EU as well as in Montenegro. In order to detect potentially infected material, surveys were carried out since 2015. During 2019, survey of Xf was conducted in places with an increased phytosanitary risk, such as the Montenegrin coast, including areas near the port of Bar and along main roads, plant production sites and urban areas, covering especially the highest risk host plants (olive trees and ornamentals plants in nurseries). The potential host plants of Xf were inspected and sampled during the period of their active growth. A total of 40 samples of suspected plant material (Olea europaea, Myrtus communis, Nerium oleander, Lavandula spp., Rosmarinus officinalis, Laurus nobilis) were collected. Symptoms on these plants varied and included necrosis and wilting of leaves, marginal leaf chlorosis and defoliation, twigs dieback. Since Xf is a vector-transmitted bacterium, five adults of Philaenus spumarius, were collected from two sites in Montenegro, and tested for the presence of Xf. Real-time loop-mediated isothermal amplification (real-time LAMP) (Harper et al., 2010; Yaseen et al., 2015) was applied for the detection of Xf in the samples of plant and insect material. LAMP system consisted of a portable ICGENE mini device (Enbiotech S.r.L., Italy) and a ready to use diagnostic kit “Xylella Screen Glow”. During the analysis, only the positive control developed a distinctive amplification curve. No positive samples were detected. The advantages of real-time LAMP method are easy handling and rapid results of the analysis. Generally this test is suitable for the detection of Xf in plant and insect vector material. Results of the 2019 survey and LAMP laboratory analyses confirmed that Xf was not present in Montenegro., ME; PPT; tamara.popovic@ubh.gov.me

Xanthomonads, members of the familyXanthomonadaceae, are economically important plant pathogenic bacteria responsible for infections of over 400 plant species. Bacteriophage-based biopesticides can provide an environmentally friendly, effective solution to control these bacteria. Bacteriophage-based biocontrol has important advantages over chemical pesticides, and treatment with these biopesticides is a minor intervention into the microflora. However, bacteriophages’ agricultural application has limitations rooted in these viruses’ biological properties as active substances. These disadvantageous features, together with the complicated registration process of bacteriophage-based biopesticides, means that there are few products available on the market. This review summarizes our knowledge of theXanthomonas-host plant and bacteriophage-host bacterium interaction’s possible influence on bacteriophage-based biocontrol strategies and provides examples of greenhouse and field trials and products readily available in the EU and the USA. It also details the most important advantages and limitations of the agricultural application of bacteriophages. This paper also investigates the legal background and industrial property right issues of bacteriophage-based biopesticides. When appropriately applied, bacteriophages can provide a promising tool against xanthomonads, a possibility that is untapped. Information presented in this review aims to explore the potential of bacteriophage-based biopesticides in the control of xanthomonads in the future.

With the increasing social concern in avoiding, or at least reducing, the application of pesticides and chemical fertilizers, in favor of sustainable eco-friendly alternatives, the search for beneficial microorganisms and microbial-derived compounds has become one of the most popular Research Topics in the field of plant-microbe interactions (Cardoso Filho, 2019; Omomowo and Babalola, 2019). Bacterial and fungal endophytes ubiquitously inhabit plant tissues without causing any adverse effect. On the contrary, their presence is often of benefit for the host, as they improve tolerance to abiotic adversities, enhance growth, and, relevantly, can modulate plant immune response and suppress pathogen colonization (Dini-Andreote, 2020). Since endophytic microorganisms typically cover the same ecological niches occupied by fungal and bacterial phytopathogens, they have been widely proposed as biocontrol agents that could be used as an alternative to pesticides (Compant et al., 2013). Thanks to the multifaceted role they play, endophytic microbial resources are now considered crucial in the perspective of their potential use to achieve sustainable improvements in the agro-food system. As a consequence, there is now a scientific ferment trying to analyze every aspect of their interaction with plants and associated pathogens. With 16 Original Research Articles and one Review, this Research Topic provides an overview of the current state of the art on the large research effort currently dedicated to understanding the role of endophytes in plant health and defense against pathogens.

The term "endophytes" usually refers to communities of microorganisms, typically bacteria or fungi, which colonize the internal portion of plants, mainly vascular tissues, interacting with the host in either beneficial or neutral mode. The scientific community customarily classifies endophytes into three main subsets based on facultative, obligate or opportunistic colonization of the host. The benefits of using fungal and bacterial endophytes in plant health are only beginning to be explored, but have become a hot topic in the search for biological control strategies. The bursting of the latest techniques of DNA high-throughput sequencing and the simultaneous development of the most diverse -omics technologies, specially metagenomics, enabled to open the horizon on a world that seems to contain a high level of biodiversity, as it includes hundreds of endophytic species per plant, many of which are unculturable. A large number of research efforts are currently dedicated to the discovery of the mechanisms by which endophytic communities can interact with the plant, to enhance growth and promote, directly or indirectly, protection from pathogens and/or insects. At a time when biocontrol has become a spellbound word, as modern policies seek an alternative and sustainable strategy to the use of traditional phytochemicals and pesticides, endophytes have become one of the most attractive biological agents in IPM programs. This Research Topic aims to gather part of the impressive number of ongoing studies dedicated to unravel the interactions between endophytes and plant pathosystems, providing an updated picture that is representative of the different branches of investigation, including: - Endophyte recognition and selection by plants. The molecular basis of endophyte-plant compatibility or incompatibility. - Endophyte dynamics and transmission between and within plants, including endophyte spread in nature, seed transmission, movement and stability of endophytes within and between plant tissues. - Metagenomic studies on the role of endophytic communities in plant health and interactions with disease-causing agents. Please note, studies using omics approaches will be considered for review only if they address mechanistic and functional insights into plant endophytes. - Mechanisms behind plant immunity triggered by fungal and bacterial endophytes. - Mechanisms of direct endophytic inhibition of plant pathogens (different from priming immune system), including secondary metabolite release, antibiosis, competition over nutrients and niche.

Bacteriophages are currently used as an alternative method for controlling bacterial spot disease on tomato incited by Xanthomonas campestris pv. vesicatoria. However, the efficacy of phage is greatly reduced due to its short residual activity on plant foliage. Three formulations that significantly increased phage longevity on the plant surface were tested in field and greenhouse trials: (i) PCF, 0.5% pregelatinized corn flour (PCF) + 0.5% sucrose; (ii) Casecrete, 0.5% Casecrete NH-400 + 0.5% sucrose + 0.25% PCF; and (iii) skim milk, 0.75% powdered skim milk + 0.5% sucrose. In greenhouse experiments, the nonformulated, PCF-, Casecrete-, and skim milk-formulated phage mixtures reduced disease severity on plants compared with the control by 1, 30, 51, and 62%, respectively. In three consecutive field trials, nonformulated phage caused 15, 20, and 9% reduction in disease on treated plants compared with untreated control plants, whereas plants treated with PCF- and Casecrete-formulated phage had 27, 32, and 12% and 30, 43, and 24% disease reduction, respectively. Plants receiving copper-mancozeb treatments were included in two field trials and had a 20% decrease in disease in the first trial and a 13% increase in the second one. Skim milk-formulated phage was tested only once and caused an 18% disease reduction. PCF-formulated phage was more effective when applied in the evening than in the morning, reducing disease on plants by 27 and 13%, respectively. The Casecrete-formulated phage populations were over 1,000-fold higher than the nonformulated phage populations 36 h after phage application.

Obradovic, A., Jones, J. B., Momol, M. T., Balogh, B., and Olson, S. M. 2004. Management of tomato bacterial spot in the field by foliar applications of bacteriophages and SAR inducers. Plant Dis. 88:736-740. Various combinations of the harpin protein, acibenzolar-S-methyl, and bacteriophages were compared for controlling tomato bacterial spot in field experiments. Harpin protein and acibenzolar-S-methyl were applied every 14 days beginning twice before transplanting and then an additional four applications throughout the season. Formulated bacteriophages were applied prior to inoculation followed by twice a week at dusk. A standard bactericide treatment, consisting of copper hydroxide plus mancozeb, was applied once prior to inoculation and then every 7 days, while untreated plants served as an untreated control. Experiments were conducted in north and central Florida fields during fall 2001, spring 2002, and fall 2002. In three consecutive seasons, acibenzolar-S-methyl applied in combination with bacteriophage or bacteriophage and harpin significantly reduced bacterial spot compared with the other treatments. However, it did not significantly affect the total yield compared with the standard or untreated control. Application of host-specific bacteriophages was effective against the bacterial spot pathogen in all three experiments, providing better disease control than copper-mancozeb or untreated control. When results of the disease severity assessments or harvested yield from the bacteriophage-treated plots were grouped and compared with the results of the corresponding nonbacteriophage group, the former provided significantly better disease control and yield of total marketable fruit.

In September 2012, soft rot symptoms on broccoli (Brassica oleracea L. var. italica Plenck) were observed in several commercial fields in the western part of Serbia. Following the first harvest, water-soaked areas developed on broccoli stem tissue and progressed into soft rot decay of entire plants. The incidence of disease was approximately 30%. In Serbia, broccoli is grown on smaller fields compared to other vegetables, but its production and consumption increased significantly in recent years. From the diseased tissue, shiny, grayish white, round colonies were isolated on nutrient agar. Six non-fluorescent, gram-negative, facultative anaerobic, oxidase-negative, and catalase-positive bacterial strains were chosen for further identification. All strains caused soft rot on potato and carrot slices and did not induce hypersensitive reaction on tobacco leaves. They grew at 37°C and in yeast salts broth medium containing 5% NaCl (2), did not produce acid from α-methyl glucoside, but utilized lactose and trehalose, and did not produce indole or lecitinase. Investigated strains formed light red, 1.5-mm-diameter colonies on Logan's medium (2), and did not produce blue pigmented indigoidine on glucose yeast calcium carbonate agar (2) nor “fried egg” colonies on potato dextrose agar. Based on biochemical and physiological characteristics (1) and ITS-PCR and ITS-RFLP analysis (4), the strains were identified as Pectobacterium carotovorum subsp. carotovorum. The 16S rRNA gene sequence from two strains (GenBank KC527051 and KC527052) showed 100% identity with sequences of P. carotovorum subsp. carotovorum previously deposited in GenBank (3). Pathogenicity of the strains was confirmed by inoculation of broccoli head tissue fragments. Three florets per strain were inoculated by pricking the petals with a syringe and hypodermic needle and depositing a droplet of bacterial suspension (approx. 1 × 108 CFU/ml) at the point of inoculation. Sterile distilled water was used as a negative control. Inoculated florets were placed in a sealed plastic container and incubated in high humidity conditions at 28°C. Tissue discoloration and soft rot developed around the inoculation point within 48 to 72 h. No symptoms developed on control florets. Identity of bacterial strains reisolated from inoculated plant tissues was confirmed by ITS-PCR using G1/L1 primers followed by digestion of PCR products with Rsa I restriction enzyme (4). In Serbia, P. carotovorum subsp. carotovorum has been isolated from potato, some vegetable crops, and ornamentals, but not from broccoli until now. References: (1) S. H. De Boer and A. Kelman. Page 56 in: Laboratory Guide for Identification of Plant Pathogenic Bacteria, 3rd ed. N. W. Schaad et al., eds. The American Phytopathological Society, St. Paul, MN, 2001. (2) P. C. Fahy and A. C. Hayward. Page 337 in: Plant Bacterial Diseases: A Diagnostic Guide. P. C. Fahy and G. J. Persley eds. Academic Press, New York, 1983. (3) S. Nabhan et al. J. Appl. Microbiol. 113: 904, 2012. (4) I. K. Toth et. al. Appl. Environ. Microbiol. 67:4070, 2001.

Bacterial spot caused by Xanthomonas euvesicatoria is one of the most devastating pepper diseases in Serbia. Questionable seed quality, climatic conditions, and frequent irrigation during summer favour the disease occurrence and spread. The available management practices do not provide adequate disease control. Therefore, development of alternative and more sustainable disease management strategies is needed. Integration of classical and biological treatments could be an effective, environmentally safe option for reducing pepper bacterial spot severity. In order to develop an efficient integrated disease management program, we studied efficacy of biocontrol agents (bacteriophage strain K Phi 1 and two strains of Bacillus subtilis AAac and QST 713), systemic acquired resistance (SAR) inducer (acibenzolar-S-methyl - ASM), a commercial microbial fertilizer (Slavol), copper based compounds (copper hydroxide and copper oxychloride) in combination with or without mancozeb, and antibiotics (streptomycin sulphate and kasugamycin). They were applied as single treatments in two separate field experiments. Based on the single treatment efficacy, various combinations of the treatments were chosen for further testing in three separate field experiments. Additionally, we evaluated potential negative effect of ASM on pepper growth and yield in the growth chamber experiment. All the tested single treatments significantly reduced disease severity compared to the inoculated control (IC), except microbiological fertilizer and the antagonistic strain AAac. Integration of copper hydroxide, ASM and bacteriophages was the most efficient treatment, reducing the disease intensity by 96-98%. The results indicated that this combination may be an adequate alternative program for control of pepper bacterial spot. This is the peer-reviewed version of the article: Šević Milan, Gašić Katarina, Ignjatov Maja, Mijatović Mirjana, Prokić Anđelka, Obradović Aleksa, "Integration of biological and conventional treatments in control of pepper bacterial spot" 119 (2019):46-51, [https://doi.org/10.1016/j.cropro.2019.01.006]

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.

The diversity of 30 Erwinia amylovora strains, isolated from quince, pear and apple trees on 14 localities in Serbia, was studied using bacteriological and molecular methods. In pathogenicity tests, all strains caused necrosis and oozing of bacterial exudate on inoculated immature pear, cherry and plum fruits, and induced hypersensitive reaction in tobacco leaves. The studied strains were Gram and oxidase negative, non-fluorescent, levan and catalase positive and facultatively anaerobic. The strains did not reduce nitrates, but utilized citrate and produced acid from sorbitol, hydrolyzed gelatine, produced reducing substances from sucrose and grew in the presence of 5% NaCl, but not at 36oC. Identity of the strains was confirmed by conventional and nested PCR methods. Rep-PCR with REP, ERIC and BOX primers resulted in amplification of several DNA fragments respectively, but showed no variation within the strains. However, different genetic profiles were obtained with RAPD-PCR by using six primers which enabled differentiation of the strains into four groups. Genetic differences between the studied strains did not correlate with the host plants, geographical origin or year of isolation. Primenom standardnih bakterioloških i molekularnih metoda proučen je diverzitet 30 sojeva Erwinia amylovora izolovanih iz dunje, kruške i jabuke, poreklom iz 14 lokaliteta u Srbiji. Svi proučavani sojevi izazvali su nekrozu i pojavu bakterijskog eksudata na nesazrelim plodovima kruške, trešnje i šljive, kao i hipersenzitivnu reakciju duvana. Proučavani sojevi bili su Gram i oksidaza negativni, fakultativno anaerobni, levan i katalaza pozitivni i nisu stvarali fluorescentni pigment na Kingovoj podlozi B. Svi sojevi hidrolizuju želatin, koriste citrate i stvaraju kiselinu iz sorbitola, proizvode redukujuće supstance iz saharoze, ne redukuju nitrate, razvijaju se u prisustvu 5% NaCl, ali ne i pri 36°C. Identitet sojeva potvrđen je konvencionalnim PCR i nested PCR metodama. Rep-PCR metodom korišćenjem REP, ERIC i BOX prajmera umnoženo je više fragmenata DNK čiji broj i veličina su se podudarali kod svih proučavanih sojeva. Za razliku od Rep-PCR, primenom RAPD-PCR metode uz korišćenje šest prajmera došlo je do izdvajanja različitih genetičkih profila i diferencijacije sojeva u četiri grupe. Genetičke razlike među proučavanim sojevima nisu bile u korelaciji sa domaćinima iz kojih su izolovani, niti sa njihovim geografskim poreklom i godinom izolacije.

Two plant-tumorigenic strains KFB 330(T) and KFB 335 isolated from galls on raspberry (Rubus idaeus) in Serbia, and a non-pathogenic strain AL51.1 recovered from a cherry plum (Prunus cerasifera) tumor in Poland, were genotypically and phenotypically characterized. Phylogenetic reconstruction based on 16S rDNA placed them within the genus Agrobacterium, with A. nepotum as their closest relative. Multilocus sequence analysis (MLSA) based on the partial sequences of atpD, glnA, gyrB, recA and rpoB housekeeping genes suggested that these three strains represent a new Agrobacterium species, that clustered with type strains of A. nepotum, A. radiobacter, "A. fabrum" and A. pusense. This was further supported by average nucleotide identity values (

The antifungal effects of thyme, cinnamon bark and clove bud essential oils (EOs) were investigated in vitro on Colletotrichum acutatum mycelial growth, conidial germination, appressoria formation, and in vivo on strawberry fruit disease incidence. All tested EOs, incorporated in potato-dextrose agar, inhibitedC. acutatum mycelial growth, and had a fungistatic effect at concentration 667 mu l/l of medium. Volatiles of cinnamon bark, thyme and clove bud EOs completely prevented conidial germination at the lowest concentrations of 1.53, 15.3 and 76.5 mu l/l of air, respectively, and disabled appressoria formation at concentration of 1.53 mu l/l of air. On inoculated strawberry fruit, thyme and cinnamon bark EO volatiles reduced anthracnose incidence at concentrations above 15.3 and 76.5 mu l/l of air, respectively. GC-FID and GC-MS analysis showed that major components of thyme EO were p-cymene, thymol, alpha-terpineol, carvacrol; cinnamon bark EO: trans- cinnameldehyde, trans-cinnamyl acetate; clove bud EO: eugenol and beta-caryophyllene. Our results suggest thatvolatiles of thyme and cinnamon bark EOs are effective against C. acutatum both in vitro and in vivo.

During the last 3 years, crown gall disease was observed in some young raspberry plantations throughout Serbia, causing considerable economic losses. Based on biochemical and physiological tests, PCR targeting the 23S rRNA gene, and 16S rRNA and recA gene sequence analysis, at least two different species were identified as causal agents of disease. Out of 14 strains isolated from raspberry tumors, 12 were identified as tumorigenic Rhizobium rhizogenes, one belonged to Agrobacterium tumefaciens genomic species G8, while the remaining strain formed a separate phylogenetic lineage within A. tumefaciens species complex, different from all known genomic species. All strains investigated harbored nopaline-type of Ti plasmid and showed identical pathogenic properties by inoculating several test plants. However, they were divided into two genetic groups based on PCR-RFLP analysis of Ti plasmid virA-virB2 region. Furthermore, total of nine unique ERIC-PCR profiles were identified among the strains studied. Although strains of R. rhizogenes exhibited similar ERIC-PCR profiles, they were differentiated into six distinct genetic groups. Based on the fact that some genetic groups were composed of strains originating from different geographic areas, it can be assumed that they have a common origin and were probably disseminated by movement of infected plant material.

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.

Serious outbreaks of grapevine crown gall disease were observed in major Serbian viticultural regions during the last five years. Tumorigenic Agrobacterium vitis was identified as a causal agent by using conventional bacteriological and molecular tests. The 36 studied strains of A. vitis showed homogeneous biochemical and physiological characteristics, but were heterogeneous in their pathogenic properties, especially on tomato and sunflower. Furthermore, genetic differences related to chromosomal and plasmid DNA were observed. The Ti plasmid of 35 strains was classified as the octopine/cucumopine (O/C) type, whereas one was classified as the vitopine (V) type. The O/C strains were further divided into O/C-1 and O/C-2 groups based on PCR analysis. Moreover, the sequence analysis of the 16S-23S rRNA ITS region provided robust and precise delineation of studied strains. Although a high level of genetic diversity in A. vitis strains from Serbia was revealed by using this approach, their genotypic relatedness with the strains from other countries suggested their common origin. Also, association between the chromosomal and plasmid DNA was determined for some phylogenetic groups and clusters.

Soft rot symptoms were observed on broccoli plants in several commercial fields in the western part of Serbia. Six strains of bacteria were isolated from diseased tissues and identified as Pectobacterium carotovorum subsp. carotovorum using conventional bacteriological and molecular methods. All strains were non-fluorescent, gram-negative, facultative anaerobes, oxidase-negative and catalase-positive, causing soft rot on potato and carrot slices and did not induce hypersensitive reaction on tobacco leaves. They grew in 5% NaCl and at 37C, did not produce acid from α-methyl glucoside, sorbitol and maltose, nor reducing substances from sucrose, but utilized lactose and trehalose, and did not produce indole or lecithinase. The investigated strains showed characteristic growth on Logan's medium and did not produce blue pigmented indigoidine on GYCA medium nor 'fried egg' colonies on PDA. The identity of strains was confirmed by ITS-PCR and ITS-RFLP analyses and by sequence analysis of the 16S rRNA gene. In a pathogenicity assay, all strains caused tissue discoloration and soft rot development on inoculated broccoli head tissue fragments. Simptomi vlažne truleži brokolija uočeni su u nekoliko komercijalnih zasada u zapadnoj Srbiji. Sojevi bakterija izolovani su iz obolelog biljnog tkiva i identifikovani kao Pectobacterium carotovorum subsp. carotovorum korišćenjem konvencionalnih bakterioloških i novijih molekularnih metoda. Svi proučavani sojevi bili su nefluorescentni, Gram-negativni, fakultativno-anaerobni, oksidaza i lecitinaza negativni, katalaza pozitivni, prouzrokujući vlažnu trulež cvetnih drški brokolija, kriški krompira i kriški mrkve, ali nisu indukovali hipersenzitivnu reakciju duvana. Svi sojevi razvijaju se pri 37C i u prisustvu 5% NaCl, ne proizvode kiselinu iz α-metil glukozida, sorbitola i maloze, niti redukujuće supstance iz saharoze, ali razlažu laktozu i trehalozu, i ne stvaraju indol. Proučavani sojevi ispoljili su karakterističan razvoj na Loganovoj diferencijalnoj podlozi, i nisu proizvodili plavi pigment na GYCA podlozi niti 'fried egg' kolonije na PDA podlozi. Identifikacija sojeva potvrđena je korišćenjem ITS-PCR i ITS-RFLP, kao i analizom sekvenci 16S rRNA gena. Prilikom izvođenja testa patogenosti, svi sojevi prouzrokovali su obezbojavanje tkiva i vlažnu trulež cvetnih drški brokolija.

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.

Bacterial spot caused by Xanthomonas euvesicatoria is considered one of the most important diseases of pepper in Serbia. In order to control this disease, copper based compounds (copper hydroxide, copper oxychloride, copper hydroxide + mancozeb, copper oxychloride + mancozeb), antibiotics (streptomycin sulfate and kasugamycin), resistance inducer (acibenzolar-S-methyl), antagonistic bacteria Bacillus subtilis (strains QST 713 and AAac), bacteriophage (strain K Phi 1) and one commercial microbial fertilizer, were tested for efficacy and compared to untreated control. The experiments were carried out in a greenhouse and climatic chamber conditions. Plants were inoculated with X euvesicatoria strain KFB 13 (10(8) CFU mL(-1)) using hand-held sprayer. Results of three experiments showed that all treatments except microbiological fertilizer significantly reduced intensity of bacterial spot symptoms compared to the untreated control. The most efficient treatment was acibenzolar-S-methyl reducing disease severity 93-97%. There was no statistically significant difference in efficacy of this treatment and copper hydroxide, copper oxychloride, copper hydroxide + mancozeb, copper oxychloride + mancozeb, streptomycin sulfate, kasugamycin and antagonistic strain QST 713 treatments. Application of antagonistic strain AAac and bacteriophage K Phi 1 significantly reduced disease intensity but less than the first group of treatments. Bacteriophage K Phi 1 (78-85% rH) was more effective in the climatic chamber compared to the efficacy (38% rH) in the greenhouse experiments.

Plants have evolved different strategies to protect themselves against pathogen infections. These strategies are classified as either passive or active, depending on whether they are constitutive barriers or triggered upon pathogen attack. Induced plant resistance is a type of active resistance and is defined as a state of enhanced defensive capacity developed by a plant when appropriately stimulated by biological agents or environmental stress. Different types of induced resistance have been defined based on differences in signaling pathways and spectra of effectiveness. First type is Systemic Acquired Resistance (SAR) which occurs in plants following activation of defense mechanisms triggered by contact with a plant pathogenic or feeding agent. SAR requires accumulation of signal molecule salicylic acid and is associated with the induction of pathogenesis-related (PR) proteins. In contrast, second type - Induced Systemic Resistance (ISR) is induced by selected strains of non-pathogenic plant growth promoting bacteria (PGPR) and regulated by jasmonic acid and ethylene. Both SAR and ISR require the function of the regulatory protein NPR1. In addition to biological agents, some chemical compounds can induce resistance of plants as well. Great progress has been made in recent years in understanding the physiological and biochemical basis of SAR and ISR, which led to their commercial use in plant protection. Biljke poseduju različite odbrambene mehanizme kojima se štite od napada patogena. Oni se mogu podeliti na mehanizme pasivne i aktivne prirode, u zavisnosti da li su već prisutni u biljci ili se aktiviraju nakon infekcije. Jedan od vidova aktivne otpornosti je indukovana otpornost koja predstavlja fiziološko stanje povećane odbrambene sposobnosti biljke i može biti izazvana različitim biotskim ili abiotskim faktorima. Na osnovu razlike u signalnim putevima i efikasnosti, definisana su dva tipa indukovane otpornosti. Prvi tip predstavlja sistemično stečena otpornost (Systemic Acquired Resistance - SAR) koja se ispoljava u svim delovima biljke kao posledica aktivacije odbrambenog mehanizma biljke u kontaktu sa organizmom koji je parazitira ili se hrani njenim delovima. Pri ovoj otpornosti dolazi do akumulacije salicilne kiseline kao signalnog molekula i povećane sinteze PR (Pathogenicity Related) proteina. Drugi tip, indukovanu sistemičnu otpornost (Induced Systemic Resistance - ISR) izazivaju nepatogene rizosferne bakterije (Plant Growth Promoting Rhizobacteria - PGPR). Ovaj tip otpornosti je regulisan jasmonskom kiselinom i etilenom i pri njenom ispoljavanju ne dolazi do sinteze PR proteina. Oba tipa otpornosti zahtevaju funkciju regulatornog NPR1 proteina. Osim bioloških agenasa i neka hemijska jedinjenja mogu indukovati otpornost biljaka. Poslednjih godina evidentan je napredak u razumevanju mehanizma dejstva ovih tipova otpornosti što je dovelo do njihove komercijalne primene u zaštiti bilja.

Forty Erwinia amylovora strains originating from different host plants and locations in Serbia and one strain from Montenegro were characterized by conventional, automated and molecular techniques. All strains were Gram-negative, nonfluorescent, facultative anaerobes, oxidase negative, levan positive, produced necrotic lesions followed by bacterial exudate on artificially inoculated immature pear fruits and caused HR on tobacco. Based on carbon source utilization, all strains tested with the Biolog system were identified as E. amylovora. Based on fatty acid profiles all tested strains clustered into three groups in which strains from north Serbia differed from strains isolated in central and south parts of the country. Restriction analysis of genomic DNA using XbaI and PFGE resulted in six different patterns differentiating the strains into six groups. Most of the investigated strains clustered in one group having the pattern type similar to Pt2 group described earlier as dominant in East Europe and the Mediterranean region. Two strains showed PFGE pattern similar to the previously described Pt3 pattern and one strain had pattern similar to Pt6. Based on size and number of the bands, new restriction patterns, assigned as Pt7, Pt8 and Pt9 were observed. PFGE results showed that the E. amylovora population in Serbia is not homogenous and was possibly introduced from different directions. This is the first characterization of E. amylovora collection of strains from Serbia using fatty acid analysis and PFGE.

Due to the difficulties in differentiation of phytopathogenic Agrobacterium spp. and lack of a standardized protocol, we carried out selection and evaluation of suitable methods based on the bacterial physiological, genetic and pathogenic properties. Strains of Agrobacterium tumefaciens, A. rhizogenes and A. vitis were differentiated using standard bacteriological and molecular methods. The biochemical and physiological tests confirmed authenticity of the strains. Two duplex PCR methods were conducted with four different primer pairs. In all strains, presence of plasmid virD2 and virC pathogenicity genes was detected. Chromosomal pehA gene was determined in A. vitis strain. Pathogenicity was confirmed on carrot slices and young plants of tomato and sunflower. Strains of A. tumefaciens and A. vitis were pathogenic on all test plants, while strain of A. rhizogenes induced characteristic symptoms only on carrot slices. The tests used in this study provided reliable discrimination between the three species and confirmed their identity as tumorigenic (Ti) Agrobacterium tumefaciens and A. vitis, and rhizogenic (Ri) A. rhizogenes. Usled poteškoća u razlikovanju vrsta roda Agrobacterium i nedostatka standardizovanog protokola izvršena je procena i odabir pogodnih metoda u cilju njihove diferencijacije na osnovu fizioloških, genetskih i patogenih odlika. Diferencirani su sojevi Agrobacterium tumefaciens, A. rhizogenes i A. vitis primenom standardnih bakterioloških i molekularnih metoda. Primenom diferencijalnih testova sojevi su ispoljili očekivane biohemijsko-fiziološke karakteristike. Izvedene su dve 'duplex' PCR metode sa 4 različita tipa prajmera. Kod proučavanih sojeva detektovano je prisustvo virD2 i virC gena patogenosti, koji se nalaze na plazmidnoj DNK bakterije. Prisustvo hromozomskog gena pehA, odgovornog za sintezu enzima poligalakturonaze, utvrđeno je kod soja A. vitis. Patogenost je proverena na kriškama mrkve i mladim biljkama paradajza i suncokreta. Sojevi A. tumefaciens i A. vitis bili su tumorogeni na svim test biljkama, dok je soj A. rhizogenes ispoljio patogenost jedino na kriškama mrkve. Na osnovu dobijenih rezultata, proučavani sojevi diferencirani su kao tumorogeni (Ti) Agrobacterium tumefaciens i A. vitis, i kao rizogeni (Ri) A. rhizogenes.