Nikolić, Ivan S., Stanković, Slaviša, Popović, Tatjana, Fira, Đorđe, Berić, Tanja, and Lozo, Jelena
Ovaj rad predstavlja opsežnu studiju koja obuhvata identifikaciju, utvrđivanje genetičkog diverziteta, filogenetskog statusa, patogenih karakteristika i biološke kontrole uzročnika bolesti lisne pegavosti šećerne repe koji je izolovan sa komercijalnih zasada na teritoriji AP Vojvodina. Kombinovanjem konvencionalnih mikrobioloških tehnika, LOPAT testova i amplifikacije specifičnih DNK fragmenata, 104 bakterijska izolata su determinisana kao Pseudomonas syringae pv. aptata. Procena genetičkog diverziteta kolekcije izolata, metodama genetičkog profilisanja i analizom sekvenci više genskih lokusa, pokazala je značajan diverzitet unutar patovara i postojanje tri različite linije infekcije. Rep-PCR je pokazao 25 različitih grupa genetičkih profila, dok je korišćenjem PFGE metode pokazano 17 različitih profila. Analiza parcijalnih sekvenci dva visoko konzervirana gena utvrdila je da postoje četiri različite alelske varijante za gyrB gen, dok su za gapA gen izdvojene tri alelske varijante. Dodatno, određivan je filogenetski status reprezentativnih izolata u okviru P. syringae kompleksa, i utvrđeno je da svi izolati pripadaju 02d filogenetskoj grupi. Ocenjivanje patogenog potencijala na osnovu utvrđivanja virulentnosti i agresivnosti reprezentativnih izolata je sprovedeno na 4 sorte šećerne repe. Veliki diverzitet u patogenim karakteristikama je pronađen među testiranim izolatima, dok su najagresivniji izolati P97 i P100 koji uzrokuju intenzitet bolesti u opsegu od 51-75%. Sorte šećerne repe, Lara i Jasmina su se pokazale rezistentnije na bolest nego sorte Marinela i Serenada. Testiranje opsega domaćina pokazalo je veliki diverzitet među izolatima, gde su izolati P21 i P23 imali najširi opseg domaćina. Utvrđivanje biološke kontrole P. syringae pv. aptata sprovedeno je na četiri sorte šećerne repe korišćenjem bakterijske kulture Bacillus amyloliquefaciens SS-12.6, pri čemu je detektovana inhibicija nekroze tkiva i do 92%. Sveukupno, dobijeni rezultati ukazuju na postojanje patogena šećerne repe, sa visokim intra-patovar diverzitetom, koji može voditi poreklo sa drugih biljaka ili može biti rezervoar infekcije ze druge biljke. Biološka kontrola izolata P.syringae pv. aptata je moguća i efikasna. This work represents a comprehensive study concerning identification, determination of genetic diversity, phylogenetic status, pathogenic characterization and biological control of causative agent of leaf spot disease on sugar beet isolated from commercial fields at the Serbian province AP Vojvodina. Combining conventional microbiological techniques, LOPAT tests and amplification of specific DNA fragments 104 isolates were determined as Pseudomonas syringae pv. aptata. Assessment of genetic polymorphism of collected isolates with genetic fingerprinting methods and Multi-locus sequence analysis showed significant intra-pathovar diversity and existing of three different lines of infection. In particular, Rep-PCR showed 25 different groups of genetic profiles, while PFGE method showed 17 different profiles. Analysis of partial sequences of two highly conserved housekeeping genes (gyrB and gapA genes) showed four and three different allelic variants, respectively. Additionally, the phylogenetic status of representative isolates within a P. syringae complex was determined and all isolates belonged to 02d phylogenetic group. The estimation of pathogenic potential by determining virulence and aggressiveness of representative isolates was conducted on four sugar beet cultivars. Large diversity of the pathogenic characteristics was found among tested isolates and the most aggressive isolates were P97 and P100 which caused disease severity in range from 51% to 75%. Cultivars Lara and Jasmina were showed much more resistant to disease then cultivars Marinela and Serenada. Host range testing showed large diversity between isolates, while isolates P21 and P23 has the broadest host range and caused disease symptoms on each tested plant species. Assessment of biological control of P. syringae pv. aptata was conducted on all four sugar beet cultivars using cell culture of Bacillus amyloliquefaciens strain SS-12.6. Inhibition of tissue necrosis was up to 92%. Overall, the obtained results point out to emerging sugar beet pathogen, with high intra-patovar diversity, which can originate from other plants or be a reservoir of infection for other plants. Biological control of isolates P. syringae pv. aptata is possible and efficient.