Identification, characterization and genetic diversity of strains of Agrobacterium spp., grapevine crown gall causal agent

Bakteriozni rak smatra se jednim od najznačajnijih i najrasprostranjenijih bakterioznih oboljenja vinove loze u svetu. Glavni prouzrokovač ovog oboljenja je vrsta Agrobacterium vitis. Tokom poslednjih nekoliko godina primećena je izražena pojava bolesti u većini vinogradarskih rejona u Srbiji. Stoga je u ovom radu proučena etiologija bakterioznog raka vinove loze u našoj zemlji. Takođe, izvršena je karakterizacija i proučen genetički diverzitet prouzrokovača bolesti. Primenjene su kako klasične bakteriološke, tako i molekularne metode. U radu su takođe korišćeni i brojni referentni sojevi A. vitis poreklom iz međunarodnih kolekcija. Simptomatični uzorci vinove loze prikupljeni su u 22 lokaliteta, raspoređena u gotovo svim vinogradarskim rejonima u našoj zemlji. Izolacija bakterija vršena je na neselektivne podloge sa manitolom i kvaščevim ekstraktom (YMA). Preliminarna identifikacija izolovanih sojeva, izvedena je primenom PCR metode, korišćenjem prajmera specifičnih za plazmidne (virC, virF, virD2, ipt, tms2) i hromozomske (pehA, 23S rRNK) gene. Među izolovanim sojevima identifikovan je tumorogeni A. vitis, a za dalji rad izdvojeno je ukupno 36 reprezentativnih sojeva. Rezultati dobijeni primenom PCR metode potkrepljeni su filogenetskom analizom zasnovanoj na parcijalnoj sekvenci 16S rRNK gena. Taksonomska pripadnost proučavanih sojeva dalje je potvrđena primenom klasičnih bakterioloških metoda. Svi proučavani sojevi ispoljili su morfološke, odgajivačke i biohemijsko-fiziološke odlike karakteristične za vrstu A. vitis. Jedini izuzetak bio je soj KFB 243, koji je i pri ponavljanju testa stvaranja kiseline iz eritritola kao izvora ugljenika ispoljio odlike svojstvene vrsti A. rhizogenes/biovar 2. U cilju provere patogenosti proučavanih sojeva, vršena je inokulacija biljaka vinove loze, suncokreta i paradajza. Od ukupno 36 proučavanih sojeva, 34 su prouzrokovala razvoj tipičnih tumora na inokulisanim biljkama vinove loze. U testu patogenosti na biljkama suncokreta i paradajza, zabeležene su značajne varijacije u intenzitetu simptoma, kako unutar pojedinačnog ogleda, tako i među različitim ponavljanjima...Crown gall is one of the most important and widespread bacterial diseases of grapevine throughout the world. It is predominantly caused by Agrobacterium vitis. During the last few years, serious outbreaks of the disease were observed in major viticultural regions in Serbia. Therefore, the objective of this research was to study etiology of the grapevine crown gall in this country. Characterization and study of genetic diversity of the pathogen were also performed, using conventional bacteriological and molecular methods. Various reference strains of A. vitis originating from international collections were also included. Symptomatic samples of grapevine were collected from 22 localities distributed in almost all viticultural regions in the country. Isolation of bacteria was performed on non-selective media with mannitol and yeast extract (YMA ). Preliminary identification of the isolated strains was performed using PCR method with primers specific for the plasmid (virC, virF, virD2, ipt, tms2) and chromosomal (pehA, 23S rRNA) genes. Among isolated strains, 36 representative tumorigenic A. vitis strains were selected for further study. Results obtained by PCR method were supported by phylogenetic analysis based on partial sequence of 16S rRNA gene. Taxonomic position of studied strains was further confirmed using conventional bacteriological methods. All the strains showed morphological, biochemical and physiological characteristics of A. vitis. The only exception was strain KFB 243, which showed characteristic of A. rhizogenes/biovar 2 in test of acid production from erythritol. Pathogenicity of the strains was studied by inoculation of grapevine, sunflower, and tomato plants. Out of 36 strains studied, 34 caused development of typical tumors on inoculated grapevine. In pathogenicity assay on tomato and sunflower plants, considerable variations in the intensity of symptoms were recorded, both within a single experiment, and among different repetitions. Therefore, use of tomato and sunflower as test plants, despite the rapid symptom development, cannot be recommended as a reliable test..

Bakteriozni rak vinove loze

Crown gall is considered one of the most important and widespread bacterial diseases of grapevine (Vitis vinifera L.). Disease is present in almost all grapevine-growing areas worldwide. Crown gall is an economically important plant disease of grapevine, especially in nurseries and newly established vineyards. Typical symptoms of grapevine crown gall disease include tumor formation on the aerial parts of host plants. Agrobacterium vitis is primary species causing this disease. A. vitis colonizes grapevine systemically and can persists latently in symptomless plant material. It is also detected as an epiphyte on grapevine and can survive in soil exclusively in association with host plants. Crown gall can be efficiently disseminated to distant geographical areas via international trade of propagation material, which is considered the main pathway of disease introduction and spread. In Serbia, crown gall was observed sporadically on grapevine, although it occurred with high incidence and severity in some years. Because there are no effective curative control measures, the disease is especially challenging to manage. Therefore, the disease management strategy is based on preventive measures. Use of pathogen-free planting material in areas with no history of the crown gall represents main prerequisite for the disease prevention. Biological control and production of resistant grape varieties are promising as future control measures.Bakteriozni rak smatra se jednim od najvažnijih i najrasprostranjenijih bakte-rioznih oboljenja vinove loze (Vitis vinifera L.). Ustanovljen je u gotovo svim zemljama gde se gaji vinova loza. Bakteriozni rak je ekonomski vrlo značajno oboljenje vinove loze, a posebno je ozbiljno u rasadnicima i mladim vinogradima. Tipični simptomi bakterioznog raka ispoljavaju se u vidu tumora na nadzemnim delovima vinove loze. Vrsta Agrobacterium vitis glavni je prouzrokovač ovog obo-ljenja. Patogen sistemično zaražava vinovu lozu i prisutan je u sprovodnom tkivu domaćina, a može biti latentno prisutan u asimptomatičnom biljnom materijalu. Takođe, patogen je detektovan i kao epifit na površini različitih organa vinove loze, kao i u rizosferi i zemljištu u blizini biljaka domaćina. A. vitis može biti raširen na velike udaljenosti putem naizgled zdravog sadnog materijala, što predstavlja najznačajniji način introdukcije i širenja bolesti. U Srbiji, bakteriozni rak se javlja sporadično, mada je u pojedinim godinama zabeležena jača pojava ovog obolje-nja uz visoku stopu zaraženih biljaka i izražene štete. Kontrola bakterioznog raka nije laka, s obzirom da ne postoje efikasne kurativne mere. Stoga se strategija zaštite prvenstveno zasniva na preventivnim merama. Korišćenje zdravog sad-nog materijala i zasnivanje vinograda na površinama koje nisu kontaminirane patogenom neizostavna je mera. Biološka kontrola i proizvodnja otpornih sorti predstavljaju obećavajuće mere kontrole u budućnosti

Populacija bakterija u bakterioznim tumorima koštičavog voća u Srbiji

Proizvodnja koštičavog voća zauzima značajno mesto u voćarstvu Srbije. Tradicionalno, najviše se gaji šljiva sa sve raznovrsnijim sortimentom u poslednjih desetak godina, dok je proizvodnja trešnje, višnje i kajsije sve više zastupljena u pojedinim regionima zemlje, nekoliko decenija unazad. Prema podacima FAO za 2021. godinu, prinos šljive bio je oko 40.000 t, a trešanja i višanja oko 15.000 t. Iste godine, proizvedeno je oko 30.000 t kajsije. Uspešnu proizvodnju često ometa pojava različitih biljnih bolesti. Od bakterioznih oboljenja, bakteriozni rak korena i korenovog različitih gajenih vrsta zauzima značajno mesto. S obzirom da je utvđeno da bakteriozne tumore osim tumorogenih, naseljavaju i druge vrste bakterija, deo istraživanja posvećen je izučavanju sastava populacije mikroorganizama tumora. Tokom 2020. i 2021. godine na području Srbije, prikupljeni su uzorci šljive, trešnje, višnje i kajsije sa izraženim simptomima bakterioznog raka. Primenom savremene metode sekvenciranja umnoženih produkata regiona V3-V4 16S rRNK gena, dobijene su amplikonske sekvence (ASV), koje su dalje obrađene. Na taj način, determinisane su najzastupljenije vrste bakterija u testiranim uzorcima. Patogena vrsta R. tumorigenes detektovana je kod trećine od ukupnog broja testiranih uzoraka. U uzorcima u kojima je prisutna, njena relativna zastupljenost iznosi do ~ 4%. Rhizobium spp. prisutan je do ~ 11% u uzorcima šljive, odnosno ~ 6% u uzorcima kajsije i ~ 5% trešnje i višnje. Vrste roda Bacillus detektovane su u svim testiranim tumorima, sa udelom i do ~ 53% kod uzoraka šljive. U tumorima na trešnji i višnji, prisutne su vrste roda Rachnella u koncentraciji do ~ 23%, kao i Cutibacterium do ~ 14%. Vrste roda Pseuodmonas detektovane su u uzrocima tumora na kajsiji (do ~4%). Osim navedenih, u uzorcima su prisutni i drugi taksoni u nižim koncentracijama. Predstavljeni rezultati su deo projekta koji se sprovodi u cilju boljeg razumevanja odnosa između patogenih i nepatogenih vrsta u zajednici mikroorganizama tumora. Imajući u vidu sve veći značaj pojave bakterioznog raka u različitim zasadima širom sveta, kao i potencjalni benefit mikroorganizama koji naseljavaju tumore u biološkoj kontroli i opštem stanju biljke domaćina, istraživanja na ovu temu će biti od značaja i u narednom periodu

Diferencijacija Pseudomonas syringae patogenih varijeteta poreklom iz koštičavih voćaka

Due to an overlapping host range, similar symptomatology and many common characteristics, Pseudomonas syringae pathovars originating from stone fruits can easily be misidentified. In order to select tests for rapid and efficient differentiation of P. s. pvs. syringae, morsprunorum and persicae, we studied the suitability and differentiating potential of some standard bacteriological and molecular methods. Differentiation of the strains was performed using LOPAT, GATTa and ice nucleation tests, nutrient sucrose broth growth and utilization of various carbon sources. PCR method enabled the detection of toxin-producing genes: syrB and syrD in P. s. pv. syringae, and cfl gene in P. s. pv. morsprunorum race 1. Syringomycin production by pv. syringae was confirmed in bioassay using Geotrichum candidum, Saccharomyces cerevisiae and Rhodotorula pilimanae as indicator organisms. Pathogenicity test on lemon and immature nectarine fruits, as well as on string bean pods, showed different intensity of reaction of the inoculated material which could separate pv. syringae from the other two pathovars. PCR-based repetitive sequences, Rep-PCR with REP, ERIC and BOX primers revealed different genetic profiles within P. syringae pathovars.Patogeni varijeteti Pseudomonas syringae poreklom sa koštičavih voćaka poseduju brojne zajedničke karakteristike u pogledu kruga domaćina, simptomatologije i biohemijskofizioloških osobina, što otežava njihovu identifikaciju. U cilju odabira testova pogodnih za brzu i pouzdanu identifikaciju P. s. pv. syringae, morsprunorum i persicae, primenjeni su standardni bakteriološki i molekularni testovi. Diferencijacija sojeva izvršena je LOPAT i GATTa testovima, posmatranjem razvoja u hranljivom rastvoru sa saharozom, sposobnošću sojeva da formiraju čestice leda, kao i mogućnošću korišćenja različitih ugljenikovih jedinjenja. PCR metod korišćen je u detekciji gena odgovornih za proizvodnju toksina siringomicina kod soja P. s. pv. syringae (syrB i syrD geni) i koronatina kod soja P. s. pv. morsprunorum rase 1 (cfl gen). Proizvodnja siringomicina potvrđena je i biotestom, korišćenjem gljiva Geotrichum candidum, Saccharomyces cerevisiae i Rhodotorula pilimanae kao indikatora. Proverom patogenosti sojeva na plodovima limuna, nesazrelim plodovima nektarine i mahunama boranije, došlo je do ispoljavanja simptoma različitog intenziteta, na osnovu kojih se može izdvojiti pv. syringae od ostala dva patovara. Primenom Rep-PCR metode, uz korišćenje REP, ERIC i BOX prajmera, ustanovljene su razlike u genetskim profilima proučavanih P. syringae patogenih varijeteta

Polyphasic study of phytopathogenic bacterial strains associated with deep bark canker of walnut in Serbia revealed a new species, Brenneria izbisi sp. nov

Serious outbreaks of walnut deep bark canker were observed on young walnut trees (Juglans regia L.) in two localities in the northern part of Serbia during 2020. From the symptomatic walnut tissues, two types of bacterial colonies were isolated, predominantly, light cream, circular and smooth colonies, as well as small, yellowish, mucoid and convex ones. PCR analysis and phenotypic assays suggested that the former group belongs to Brenneria spp., while the latter isolates were identified as Xanthomonas arboricola pv. juglandis. Within the Brenneria group, two strains were identified as Brenneria nigrifluens, while other 15 strains did not belong to any Brenneria species described so far. Therefore, we selected four representative strains of the unknown Brenneria sp. and subjected them to polyphasic analysis. As expected, in a phylogenetic tree based on partial 16S rDNA sequences, four novel strains grouped with other Brenneria representatives, and showed close phylogenetic relationship to Brenneria salicis. Furthermore, multilocus sequence analysis (MLSA) based on the partial sequences of atpD, gyrB, infB and rpoB housekeeping genes and core-genome phylogeny indicated that the studied strains form a novel and a clearly separate Brenneria lineage. Overall genome relatedness indices showed that they represent a new Brenneria species. The new species can be differentiated from the other Brenneria spp. infecting walnut and closely related B. salicis strains based on phenotypic characteristics, as well. Moreover, the pathogenicity tests on two-year-old walnut plants proved the ability of strains to cause necrosis and longitudinal black lesions and cracks on the trunk and branches of walnut trees. Overall, polyphasic characterization showed that the studied strains isolated from walnut with symptoms of deep bark canker represent a novel species of the genus Brenneria for which the name Brenneria izbisi sp. nov. is proposed. The type strain of B. izbisi is KBI 423T (= CFBP 9035T = LMG 32479T). To facilitate rapid identification of newly described species, a conventional PCR protocol and primers targeting the putative gene hrpP, were developed. Further study should reveal the potential role of each pathogen isolated from symptomatic walnut in disease development as well as possible interaction between them

Specificity and sensitivity of three pcr-based methods for detection of erwinia amylovora in pure culture and plant material

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

BIOCTA: Novel approach to biocontrol of recently described plant tumorogenic Rhizobium spp. using autochthonous microbial solutions

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

PSEUDOMONAS SPP. IN BIOCONTROL OF CROWN GALL DISEASE: NEW APPROACHES

INTRODUCTION Crown gall is an economically important and widespread plant disease caused by tumorigenic bacteria that are commonly affiliated with the genera Agrobacterium, Allorhizobium and Rhizobium. Novel and an atypical group of tumorigenic agrobacteria belonging to the genus Rhizobium (“tumorigenes” clade) was identified as a causative agent of crown/cane gall on blackberry, rhododendron and blueberry in Serbia and Germany (Kuzmanović et al., 2018 and 2019). Efficient measures to control crown gall disease were not reported till nowadays, so assessment and application of alternative biological control measures would contribute to sustainable agricultural production and environmental protection. The aims of the study were 1) identification of candidate bacterial strains that could be employed for biological control 2) to analyse phytobiome of the treated and non-treated crops and 3) to perform a whole-genome sequencing of a few most promising biocontrol strains. STATE OF THE ART Antimicrobial activity of ten biocontrol candidates from rhododendron and 27 additional antagonistic strains were tested in vitro against the tumor-inducing strain Rhizobium sp. rho-6.2. The six most efficient Pseudomonas and Bacillus strains were tested in vivo, using co-inoculation and preventive inoculation strategies in controlled greenhouse conditions on tomato plants as a model system in four replicas and randomized. Tumors from the most effective treatments were sampled, and then total DNA was isolated and subjected to the next-generation sequencing (NGS). Direct analysis of bacterial communities using Illumina MiSeq sequencing of 16S rRNA gene amplicon libraries was performed to assess the microbial ecological effect, with complete bioinformatic and computational biology analysis conducted. Also, a whole-genome sequencing of a few most promising antagonistic strains was performed. RESULTS Among six antagonistic strains, the most efficient in co-inoculation strategy against pathogenic Rhizobium sp. rho-6.2 were two Pseudomonas strains (R-6.10 and R-11.20), which reduced a tumor size 92.86%. The same Pseudomonas strains were less effective in preventive treatments (15.38 and 30.77%). Although Bacillus strains exhibited high in vitro antimicrobial activity, their in vivo activity was in preventive treatment only 15.38%, whilst in co-inoculation strategy was detected as moderate (42.86%). Bacillus and Pseudomonas strains applied together increased biocontrol activity with 38.6% of tumor’s reduction. In analyzed treatments, was detected the dominant presence of Proteobacteria followed by a moderate presence of Actinobacteriota and Firmicutes. On the genus level, the most abundant, both in negative control and treatments, were representatives of Allorhizobium-Neorhizobium-Pararhizobium-Rhizobium group (18,53% - 71,81%) followed by Pseudomonas spp. (2,76%- 36,46%). According to alpha diversity indexes on the genus level, the highest values were detected in the negative control, pre-treatment with Pseudomonas sp. R-6.10, co-inoculation with Pseudomonas spp. R-6.10 and R-11.20 individually. Analysis of beta diversity by the DPCoA matrix exhibited that the co-inoculation and positive control groups were well separated, whilst preventive treatment overlapped both the co-inoculation and positive control samples. Differential abundance analysis on a genus level revealed a statistically higher presence of Stenotrophomonas and Asanoa in preventive treatments and Dyadobacter and Pandoraea spp. in their positive control. In the co-inoculation strategy, Pseudolabrys and Asanoa were prevalent in treatments and Allorhizobium-Neorhizobium-Pararhizobium-Rhizobium was detected as prevalent in positive control. Whole-genome sequencing and preliminary comparative genomics analyses revealed that the best biocontrol candidates, Pseudomonas strains R-6.10 and R-11.20 represent two new species, most closely related to P. graminis and P. fildesensis, respectively. DISCUSSION The Pseudomonas species exhibited the most prominent activity in vivo. Pseudomonas genus is rich in species with the potential for biocontrol of wide spectra of pathogens. Their activity is based on the production of variety of antimicrobial compounds (Dimkic et al., 2022). Also, silencing quorum sensing or quorum quenching is one of their biocontrol strategies by attenuating the virulence of the pathogen (Zhang et al., 2021). Metabarcoding analysis showed differences between treatments, mainly on the level of less presented genera. Best candidates for biocontrol of crown gall, Pseudomonas spp. R-6.10 and R-11.20 originating from the crown gall tumor, confirms the previously established hypothesis that plants are the best sources of biocontrol agents (Janisiewicz et al., 2013). CONCLUSIONS The selected Pseudomonas strains could be further tested as an alternative strategy for the biocontrol of crown gall disease and the potential involvement of the quorum quenching mechanism will be determined. Crown gall tumors have shown to be a great source of antagonistic isolates Pseudomonas sp. R-6.10 and Pseudomonas sp. R-11.20 identified according to WGS as the two new species that further needs to be described

Karakterizacija i diverzitet populacije sojeva Erwinia amylovora poreklom iz jabučastih voćaka gajenih u Srbiji

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

Draft Genome Sequences of Agrobacterium nepotum Strain 39/7(T) and Agrobacterium sp. Strain KFB 330

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