Diferencijacija fitopatogenih vrsta roda Agrobacterium

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

61 Analiza masnih kiselina sojeva Erwinia amylovora iz Srbije i Crne Gore

Automated method of fatty acid analysis was used to identify and study heterogeneity of 41 Erwinia amylovora strains, originating from 8 plant species grown in 13 locations in Serbia and one in Montenegro. All strains contained 14:0 3OH fatty acid, characteristic for the “amylovora” group. According to fatty acid composition 39 strains were identified as E. amylovora as the first choice from the database. Due to their specific fatty acid composition, two strains were identified as E. amylovora, but as a second choice. Fatty acid analysis also showed that E. amylovora population from Serbia could be differentiated in three groups, designated in this study as α, β and γ. All strains originating from central or south Serbia, as well as four strains from north Serbia clustered into group α. Group β and γ contained only strains isolated in northern Serbia (Vojvodina). The results show that E. amylovora population in this area is heterogeneous and indicate pathogen introduction from different directions. Fatty acid analysis enabled identification at species level, as well as new insights of heterogeneity of E. amylovora population.Automatizovana metoda analize masnih kiselina primenjena je za identifikaciju i proučavanje heterogenosti Erwinia amylovora. Kao materijal za analizu prikupljen je 41 soj E. amylovora izolovan iz 8 različitih vrsta domaćina gajenih u 13 lokaliteta u Srbiji i jednom lokalitetu u Crnoj Gori. Rezultati ukazuju da svi proučavani sojevi poseduju 14:0 3OH masnu kiselinu, koja je karakteristična za „amylovora“ grupu. Na osnovu sastava masnih kiselina 39 sojeva je identifikovano kao E. amylovora, kao prvi izbor iz baze podataka. Dva soja su identifikovana kao E. amylovora, ali tek kao drugi izbor iz baze podataka, što je najverovatnije posledica specifičnosti u sastavu njihovih masnih kiselina. Rezultati analize masnih kiselina takođe pokazuju da populacija E. amylovora poreklom iz Srbije nije homogena i da među sojevima postoje tri grupe ili profila, koji su u ovom radu obeleženi sa α, β i γ. Svi sojevi koji su izolovani na prostoru centralne ili južne Srbije pripadaju grupi α, kao i četiri soja izolovana na području Vojvodine. Grupama β i γ pripadaju samo sojevi izolovani na području Vojvodine. Dobijeni rezultati predstavljaju dokaz heterogenosti populacije E. amylovora na ovim prostorima i ukazuju na mogućnost prodora patogena u naše područje iz različitih pravaca. Analiza masnih kiselina omogućila je ne samo identifikaciju do nivoa vrste, već i nova saznanja o heterogenosti populacije E. amylovora na ovim prostorima

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

Differentiation of Pseudomonas syringae Pathovars Originating from Stone Fruits

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 ofsome standard bacteriological and molecular methods. Differentiation of the strains wasperformed using LOPAT, GATTa and ice nucleation tests, nutrient sucrose broth growthand utilization of various carbon sources. PCR method enabled the detection of toxin-producinggenes: syrB and syrD in P. s. pv. syringae, and cfl gene in P. s. pv. morsprunorum race1. Syringomycin production by pv. syringae was confirmed in bioassay using Geotrichumcandidum, 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 withREP, ERIC and BOX primers revealed different genetic profiles within P. syringae pathovars

Refining the taxonomy of the order <i>Hyphomicrobiales (Rhizobiales)</i> based on whole genome comparisons of over 130 type strains

Supplementary Material for ‘Refining the taxonomy of the order Hyphomicrobiales (Rhizobiales) based on whole genome comparisons of over 130 type strains’, as described in International Journal of Systematic and Evolutionary Microbiology</p