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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.

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.

Tokom 2007. godine zabeležena je masovna pojava bakteriozne plamenjače jabučastih voćaka prouzrokovana bakterijom Erwinia amylovora. Pored nekroze plodova i plamenjače mladara, tipičnih simptoma bolesti, našu pažnju je privukla pojava nekroze mrkoljubičaste boje u zoni korenovog vrata, koja prstenasto zahvata prizemni deo i prouzrokuje izumiranje stabala jabuke. S obzirom na to da uzrok ovakvih patoloških promena može da bude E. amylovora, ali i fitopatogene gljive iz rodova Phomopsis i Phytophthora, preduzeta su istraživanja sa ciljem da se utvrdi etiologija ovog oboljenja jabuke. Iz obolelih uzoraka izolovane su bakterije koje stvaraju levan, ali ne i fluorescentni pigment. Proučavani sojevi prouzrokovali su HR na listovima duvana i nekrozu inokulisanih, nesazrelih plodova kruške, uz obilno stvaranje bakterijskog eksudata, što je karakterističan znak E. amylovora. Na osnovu rezultata dobijenih pri testovima provere patogenosti, ali i rezultata biohemijskih odlika, ELISA testa i molekularnih metoda (PCR), potvrđeno je da proučavani izolati pripadaju bakteriji E. amylovora, koja je uzročnik simptoma nekroze korenovog vrata stabala jabuke, prvi put zabeleženih u Srbiji. Moguće objašnjenje ove pojave je da su vegetativne podloge za kalemljenje bile inficirane ovom bakterijom. Zbog toga je veoma bitno razvijati dijagnostičke protokole za utvrđivanje prisustva E. amylovora u podlogama za kalemljenje jabuke, tokom procesa kontrole zdravstvene ispravnosti sadnog materijala., A large-scale outbreak of fire blight symptoms caused by Erwinia amylovora was recorded in pome fruit trees during 2007. In addition to fruit necrosis and shoot blight as the typical disease symptoms, dark purple necrosis was observed in the root collar area girdling the trunk just above the ground and thus withering the whole apple tree. Since similar symptoms on apple trees could be caused by E. amylovora or one of several phytopathogenic fungi of the genera Phomopsis and Phytophthora, an investigation was conducted to identify the causal agent of this disease. Levan-producing, nonfluorescent bacteria were isolated from diseased samples. The isolated strains produced HR in tobacco leaves and necrosis of artificially inoculated, immature pear fruits, followed by oozing of bacterial exudate, a characterisitic of E. amylovora. Based on the results of pathogenicity tests, biochemical characteristics, ELISA test and PCR analysis, it was confirmed that the investigated strains belonged to E. amylovora, causing the root collar necrosis of apple trees as an atypical symptom of this bacterium in Serbia. The explanation of this symptom may be that the vegetative rootstocks were infected with E. amylovora. Therefore, the development of diagnostic protocols for detection of E. amylovora in apple rootstock is very important for health inspections of planting materials.

Bacterial strains were isolated from above- and underground parts of diseased calla plants originating from different localities in Serbia and one locality in Montenegro. They were characterized by studying their pathogenic, cultural, biochemical and physiological characteristics. All investigated strains caused soft rot of calla leaf stalks, potato slices and aloe leaves, and induced hypersensitive reaction on tobacco. Bacteriological properties of the strains indicated that symptoms on calla plants were caused by Gram-negative, nonfluorescent, oxidase negative, catalase positive and facultatively anaerobic bacterium belonging to the genus Pectobacterium. The investigated strains grew at 37ºC and in 5% NaCl, utilised lactose and trechalose, and produced neither indol nor lecitinase. These results, as well as the characteristic growth on Logan's differential medium indicated that soft rot of tuber and stem base of calla plants was caused by Pectobacterium carotovorum subsp. carotovorum. This is the first report of this pathogen affecting calla plants in Serbia., Proučene su patogene, odgajivačke i biohemijsko-fiziološke odlike sojeva izolovanih iz nadzemnih i podzemnih delova obolelih biljaka kale gajene u različitim lokalitetima u Srbiji i jednom lokalitetu u Crnoj Gori. Svi proučavani sojevi ispoljili su izraženu pektolitičku aktivnost prouzrokujući vlažnu trulež lisnih drški kale, kriški krompira, liski aloje ili sanseverije, a prouzrokovali su i hipersenzitivnu reakciju duvana. Proučavanjem bakterioloških karakteristika utvrđeno je da promene na biljkama kale prouzrokuju Gram-negativni, nefluorescentni, oksidaza-negativni, katalaza-pozitivni i fakultativno-anaerobni sojevi bakterije, koja prema navedenim karakteristikama pripada rodu Pectobacterium. Proučavani sojevi se razvijaju pri 37ºC i u prisustvu 5% NaCl, razlažu laktozu i trehalozu, ne stvaraju indol i lecitinazu. Navedeni rezultati, kao i karakterističan razvoj na Loganovoj diferencijalnoj podlozi ukazuju da je vlažnu trulež korena i prizemnog dela biljaka kale prouzrokovala bakterija Pectobacterium carotovorum subsp. carotovorum. Ovo je prvo saopštenje o pojavi P. c. ssp. carotovorum kao patogena kale u Srbiji.

Tokom proleća 2003. godine, zapaženi su tumori na korenu i korenovom vratu mladih biljaka maline sorte Vilamette u okolini Valjeva. Iz obolelih uzoraka izolovane su fitopatogene bakterije. Proučavanjem patogenih, morfoloških i diferencijalnih biohemijsko-fizioloških karakteristika izolovanih sojeva, utvrđeno je da proučavani sojevi pripadaju bakteriji Agrobacterium radiobacter (biovar 1). U cilju potvrde identiteta izolovanih sojeva lančanom reakcijom polimeraze (PCR), korišćeni su prajmeri komplementarni tms2 genu lociranom na Ti plazmidu. U prvoj PCR reakciji izvedenoj korišćenjem tms2F1 + tms2R2 para prajmera, detektovani su fragmenti nukleinske kiseline veličine 617 baznih parova (bp), specifični za tumorogene sojeve A. tumefaciens. U drugoj PCR reakciji korišćenjem para prajmera tms2F1 i tms2B su amplifikovani PCR produkti očekivane veličine 458 bp, karakteristični za ovu bakteriju. Na osnovu multiplex PCR reakcije korišćenjem prajmera komplementarnih na hromozomalni gen koji kodira 23S rRNA sojevi izolovani iz tumora na korenu maline svrstani su u biovar 1 Agrobacterium (A. radiobacter). Ovo je prvi nalaz ove bakterije na malini u Srbiji., During the spring of 2003, gall symptoms on the roots and crowns of young raspberry plants cv. Vilamette were observed near Valjevo. Phytopathogenic bacteria were isolated from diseased plant samples. Based on the pathogenic, morphological, differential biochemical and physiological characteristics, the isolated strains were identified as tumorigenic Agrobacterium radiobacter (biovar 1 Agrobacterium). In order to confirm the identity of isolated strains by polymerase chain reaction (PCR) primers complementary to tms2 gene located on the Ti plasmid were used. In the first PCR protocol using a tms2F1 + tms2R2 primer pair, 617 bp products specific for tumorigenic Agrobacterium strains were amplified. The second PCR protocol, using a tms2F1 + tms2B primer pair, amplified the expected 458 bp products. On the basis of multiplex PCR with primers complementary to chromosomal gene coding for 23S rRNA, the isolated strains were classified as biovar 1 Agrobacterium (A. radiobacter). This is the first report of tumorigenic A. radiobacter on raspberry in Serbia.