Obradović, Mina

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Isolation, Characterization and Draft Genome Analysis of Bacteriophages Infecting Acidovorax citrulli

Gašić, Katarina; Obradović, Mina; Kuzmanović, Nemanja; Zlatković, Nevena; Ivanović, Milan; Ristić, Lela; Obradović, Aleksa

(Frontiers Media S.A., 2022)

TY  - JOUR
AU  - Gašić, Katarina
AU  - Obradović, Mina
AU  - Kuzmanović, Nemanja
AU  - Zlatković, Nevena
AU  - Ivanović, Milan
AU  - Ristić, Lela
AU  - Obradović, Aleksa
PY  - 2022
UR  - https://plantarum.izbis.bg.ac.rs/handle/123456789/725
AB  - Bacterial fruit blotch and seedling blight, caused by Acidovorax citrulli, is one of the most destructive diseases of melon and watermelon in many countries. Pathogen-free seed and cultural practices are major pillars of the disease control. However, use of bacteriophages as natural biocontrol agents might also contribute to the disease management. Therefore, we isolated 12 bacteriophages specific to A. citrulli, from phyllosphere and rhizosphere of diseased watermelon plants. The phage strains were characterized based on their host range, plaque and virion morphology, thermal inactivation point, adsorption rate, one step growth curve, restriction fragment length polymorphism (RFLP), and genomic analysis. Transmission electron microscopy of three phage strains indicated that they belong to the order Caudovirales, family Siphoviridae. All phages lysed 30 out of 32 tested A. citrulli strains isolated in Serbia, and did not lyse other less related bacterial species. They produced clear plaques, 2 mm in diameter, on bacterial lawns of different A. citrulli strains after 24 h of incubation. The thermal inactivation point was 66 or 67°C. They were stable at pH 5–9, but were sensitive to chloroform and inactivated in either 5 or 10 min exposure to ultraviolet (UV) light. RFLP analysis using EcoRI, BsmI and BamHI enzymes did not show genetic differences among the tested phages. Adsorption rate and one step growth curve were determined for the Acidovorax phage ACF1. Draft genome sequence of the ACF1 phage was 59.377 bp in size, with guanine-cytosine (GC) content 64.5%, including 89 open reading frames. This phage shared a very high genomic identity with Acidovorax phage ACPWH, isolated in South Korea. Evaluation of systemic nature of ACF1 strain showed that it can be absorbed by roots and translocated to upper parts of watermelon plants where it survived up to 10 days.
PB  - Frontiers Media S.A.
T2  - Frontiers in Microbiology
T1  - Isolation, Characterization and Draft Genome Analysis of Bacteriophages Infecting Acidovorax citrulli
IS  - 803789
VL  - 12
DO  - 10.3389/fmicb.2021.803789
ER  - 
@article{
author = "Gašić, Katarina and Obradović, Mina and Kuzmanović, Nemanja and Zlatković, Nevena and Ivanović, Milan and Ristić, Lela and Obradović, Aleksa",
year = "2022",
abstract = "Bacterial fruit blotch and seedling blight, caused by Acidovorax citrulli, is one of the most destructive diseases of melon and watermelon in many countries. Pathogen-free seed and cultural practices are major pillars of the disease control. However, use of bacteriophages as natural biocontrol agents might also contribute to the disease management. Therefore, we isolated 12 bacteriophages specific to A. citrulli, from phyllosphere and rhizosphere of diseased watermelon plants. The phage strains were characterized based on their host range, plaque and virion morphology, thermal inactivation point, adsorption rate, one step growth curve, restriction fragment length polymorphism (RFLP), and genomic analysis. Transmission electron microscopy of three phage strains indicated that they belong to the order Caudovirales, family Siphoviridae. All phages lysed 30 out of 32 tested A. citrulli strains isolated in Serbia, and did not lyse other less related bacterial species. They produced clear plaques, 2 mm in diameter, on bacterial lawns of different A. citrulli strains after 24 h of incubation. The thermal inactivation point was 66 or 67°C. They were stable at pH 5–9, but were sensitive to chloroform and inactivated in either 5 or 10 min exposure to ultraviolet (UV) light. RFLP analysis using EcoRI, BsmI and BamHI enzymes did not show genetic differences among the tested phages. Adsorption rate and one step growth curve were determined for the Acidovorax phage ACF1. Draft genome sequence of the ACF1 phage was 59.377 bp in size, with guanine-cytosine (GC) content 64.5%, including 89 open reading frames. This phage shared a very high genomic identity with Acidovorax phage ACPWH, isolated in South Korea. Evaluation of systemic nature of ACF1 strain showed that it can be absorbed by roots and translocated to upper parts of watermelon plants where it survived up to 10 days.",
publisher = "Frontiers Media S.A.",
journal = "Frontiers in Microbiology",
title = "Isolation, Characterization and Draft Genome Analysis of Bacteriophages Infecting Acidovorax citrulli",
number = "803789",
volume = "12",
doi = "10.3389/fmicb.2021.803789"
}
Gašić, K., Obradović, M., Kuzmanović, N., Zlatković, N., Ivanović, M., Ristić, L.,& Obradović, A.. (2022). Isolation, Characterization and Draft Genome Analysis of Bacteriophages Infecting Acidovorax citrulli. in Frontiers in Microbiology
Frontiers Media S.A.., 12(803789).
https://doi.org/10.3389/fmicb.2021.803789
Gašić K, Obradović M, Kuzmanović N, Zlatković N, Ivanović M, Ristić L, Obradović A. Isolation, Characterization and Draft Genome Analysis of Bacteriophages Infecting Acidovorax citrulli. in Frontiers in Microbiology. 2022;12(803789).
doi:10.3389/fmicb.2021.803789 .
Gašić, Katarina, Obradović, Mina, Kuzmanović, Nemanja, Zlatković, Nevena, Ivanović, Milan, Ristić, Lela, Obradović, Aleksa, "Isolation, Characterization and Draft Genome Analysis of Bacteriophages Infecting Acidovorax citrulli" in Frontiers in Microbiology, 12, no. 803789 (2022),
https://doi.org/10.3389/fmicb.2021.803789 . .
3
3
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KARAKTERIZACIJA BAKTERIOFAGA SPECIFIČNIH PREMA Acidovorax citrulli – PROUZROKOVAČU MRLJAVOSTI PLODA LUBENICE

Gašić, Katarina; Obradović, Mina; Blagojević, Nevena; Obradović, Aleksa

(Društvo za zaštitu bilja, 2021)

TY  - CONF
AU  - Gašić, Katarina
AU  - Obradović, Mina
AU  - Blagojević, Nevena
AU  - Obradović, Aleksa
PY  - 2021
UR  - https://plantarum.izbis.bg.ac.rs/handle/123456789/1279
AB  - Acidovorax citrulli, prouzrokovač mrljavosti plodova lubenice i dinje jedna je od
najdestruktivnijih bakterioza ovih biljaka u svetu. Patogen je detektovan u Srbiji 2014. godine,
kada je izazvao ogromne štete proizvodnji lubenice. Usled nedostatka efikasnih baktericida za
suzbijanje ovog patogena, nameće se potreba pronalaženja alternativnih rešenja. Stoga je
proučeno prisustvo bakteriofaga, prirodnih neprijatelja Acidovorax citrulli i izvršena njihova
izolacija i karakterizacija.
Tokom 2014. godine izolovano je dvanaest sojeva faga iz rizosfere obolelih biljaka
lubenice, kako i iz listova lubenice sa simptomima bakteriozne mrljavosti. Karakterizacija
sojeva izvršena je na osnovu spektra domaćina virusa, morfoloških odlika plakova i virusnih
čestica, termalne tačke inaktivacije, parametara umnožavanja faga, kao i RFLP analize.
Rezultati istraživanja pokazali su da svi sojevi faga liziraju 30 od ukupno 32 testirana
soja A. citrulli izolovana u Srbiji. Fagi su ispoljili vosoku specifičnost prema vrsti i nisu lizirali
ostale testirane vrste patogenih i saprofitnih bakterija. Svi sojevi formirali su prozračne
plakove, prečnika 2 mm, nakon 24 h inkubacije. Pomoću transmisionog elektronskog
mikrosopa, utvrđeno je da fagi pripadaju redu Caudovirales, familiji Siphoviridae. Tokom 20
min 90% faga se adsorbuje na površinu bakterijske ćelije, dok se po jednoj inficiranoj ćeliji
oslobodi prosečno 74 ± 5 čestice virusa. Termalna tačka inaktivacije faga iznosila je 66 ili
67ºC. Fagi su ispoljili osetljivost prema hloroformu, bili su stablini pri pH 5-9, ali inaktivirani
nakon 5-10 min izloženosti UV svetlu. RFLP analizom, korišćenjem EcoRI, BsmI and BamHI
enzima nisu utvrđene genetičke razlike među proučavanim sojevima faga.
Ova istraživanja stvaraju osnovu za dalji razvoj inovativnih metoda za kontrolu
bakteriozne mrljavosti lubenice primenom prirodnih mehanizama zaštite.
PB  - Društvo za zaštitu bilja
C3  - XVI Savetovanje o zaštiti bilja, 22-25. februar 2021., Zlatibor
T1  - KARAKTERIZACIJA BAKTERIOFAGA SPECIFIČNIH PREMA Acidovorax citrulli – PROUZROKOVAČU MRLJAVOSTI PLODA LUBENICE
IS  - 17
ER  - 
@conference{
author = "Gašić, Katarina and Obradović, Mina and Blagojević, Nevena and Obradović, Aleksa",
year = "2021",
abstract = "Acidovorax citrulli, prouzrokovač mrljavosti plodova lubenice i dinje jedna je od
najdestruktivnijih bakterioza ovih biljaka u svetu. Patogen je detektovan u Srbiji 2014. godine,
kada je izazvao ogromne štete proizvodnji lubenice. Usled nedostatka efikasnih baktericida za
suzbijanje ovog patogena, nameće se potreba pronalaženja alternativnih rešenja. Stoga je
proučeno prisustvo bakteriofaga, prirodnih neprijatelja Acidovorax citrulli i izvršena njihova
izolacija i karakterizacija.
Tokom 2014. godine izolovano je dvanaest sojeva faga iz rizosfere obolelih biljaka
lubenice, kako i iz listova lubenice sa simptomima bakteriozne mrljavosti. Karakterizacija
sojeva izvršena je na osnovu spektra domaćina virusa, morfoloških odlika plakova i virusnih
čestica, termalne tačke inaktivacije, parametara umnožavanja faga, kao i RFLP analize.
Rezultati istraživanja pokazali su da svi sojevi faga liziraju 30 od ukupno 32 testirana
soja A. citrulli izolovana u Srbiji. Fagi su ispoljili vosoku specifičnost prema vrsti i nisu lizirali
ostale testirane vrste patogenih i saprofitnih bakterija. Svi sojevi formirali su prozračne
plakove, prečnika 2 mm, nakon 24 h inkubacije. Pomoću transmisionog elektronskog
mikrosopa, utvrđeno je da fagi pripadaju redu Caudovirales, familiji Siphoviridae. Tokom 20
min 90% faga se adsorbuje na površinu bakterijske ćelije, dok se po jednoj inficiranoj ćeliji
oslobodi prosečno 74 ± 5 čestice virusa. Termalna tačka inaktivacije faga iznosila je 66 ili
67ºC. Fagi su ispoljili osetljivost prema hloroformu, bili su stablini pri pH 5-9, ali inaktivirani
nakon 5-10 min izloženosti UV svetlu. RFLP analizom, korišćenjem EcoRI, BsmI and BamHI
enzima nisu utvrđene genetičke razlike među proučavanim sojevima faga.
Ova istraživanja stvaraju osnovu za dalji razvoj inovativnih metoda za kontrolu
bakteriozne mrljavosti lubenice primenom prirodnih mehanizama zaštite.",
publisher = "Društvo za zaštitu bilja",
journal = "XVI Savetovanje o zaštiti bilja, 22-25. februar 2021., Zlatibor",
title = "KARAKTERIZACIJA BAKTERIOFAGA SPECIFIČNIH PREMA Acidovorax citrulli – PROUZROKOVAČU MRLJAVOSTI PLODA LUBENICE",
number = "17"
}
Gašić, K., Obradović, M., Blagojević, N.,& Obradović, A.. (2021). KARAKTERIZACIJA BAKTERIOFAGA SPECIFIČNIH PREMA Acidovorax citrulli – PROUZROKOVAČU MRLJAVOSTI PLODA LUBENICE. in XVI Savetovanje o zaštiti bilja, 22-25. februar 2021., Zlatibor
Društvo za zaštitu bilja.(17).
Gašić K, Obradović M, Blagojević N, Obradović A. KARAKTERIZACIJA BAKTERIOFAGA SPECIFIČNIH PREMA Acidovorax citrulli – PROUZROKOVAČU MRLJAVOSTI PLODA LUBENICE. in XVI Savetovanje o zaštiti bilja, 22-25. februar 2021., Zlatibor. 2021;(17)..
Gašić, Katarina, Obradović, Mina, Blagojević, Nevena, Obradović, Aleksa, "KARAKTERIZACIJA BAKTERIOFAGA SPECIFIČNIH PREMA Acidovorax citrulli – PROUZROKOVAČU MRLJAVOSTI PLODA LUBENICE" in XVI Savetovanje o zaštiti bilja, 22-25. februar 2021., Zlatibor, no. 17 (2021).