Uporaba molekularnih metod za določanje fitopatogenih bakterij na primeru Erwinia amylovora (Burrill) Winslow et al.

Tina DEMŠAR, Nataša PETROVIČ, Dejan ŠTEBIH, Tanja DREO, Aleš Blatnik in Maja RAVNIKAR

Nacionalni inštitut za biologijo, Oddelek za rastlinsko fiziologijo in biotehnologijo, SI-1000 Ljubljana, Slovenija

Bakterijski hrušev ožig je karantenska bolezen sadnega drevja in nekaterih okrasnih rastlin. Bolezen je zastopana že skoraj po vseh državah Evrope, tudi pri naših sosedih, zato njen morebitni vnos predstavlja resno grožnjo za pridelavo hrušk, jabolk in drugega sadilnega materiala.

Postopek laboratorijskega testiranja, ki ga predpisuje organizacija EPPO (European Plant Protection Organization), temelji na izolaciji bakterije Erwinia amylovora na semiselektivnih in neselektivnih gojiščih, seroloških tehnikah (aglutinacija in imunofluorescenčni test) in testu patogenosti na nezrelih plodovih hrušk. Kot dopolnilo klasičnim laboratorijskim testom smo uvedli molekularno metodo PCR (polimerazna verižna reakcija) za potrditev izolirane bakterijske kulture, ki hkrati omogoča razlikovanje od bakterije Pseudomonas syringae pv. syringae, povzročiteljice bolezni z zelo podobnimi simptomi, in od drugih pogostih bakterij sadnega drevja.

V prispevku predstavljamo rezultate testov PCR z uporabo različnih začetnih oligonukleotidov, ki specifično pomnožujejo fragmente bakterijske DNK, in njihovo uporabnost pri rutinskem testiranju.

ABSTRACT

The use of molecular biology methods for detection of  phytopathogenic bacteria: the study case of  Erwinia amylovora (Burrill) Winslow et al.

Fireblight is a quarantine bacterial disease on fruit trees and ornamental plants recorded in most European countries. The fact that fireblight is present in all countries neighbouring Slovenia represents a very high possibility of it's introduction to Slovenia, which is a serious treat to Slovenian fruit growing, especially production of pears and apples.

We have introduced a laboratory testing scheme for a reliable detection of Erwinia amylovora (Ea), the bacterial agent causing fireblight disease. A testing procedure reccomended by EPPO (European Plant Protection Organization) includes: isolation of Ea by plating on semiselective and non-selective media, serological methods (agglutination and immunofluorescence) and pathogenicity test on young pear fruits. As an addition to all these classical laboratory tests we have introduced a molecular biology based method, which both confirms the Ea in a bacterial culture, and enables to distinguish Ea from other bacteria such as Pseudomonas syringae pv. syringae, a causal agent of a disease similar to fireblight regarding the symptoms, and from other frequently present bacteria on fruit trees.

The results of PCR testing with the use of different primers, which amplify bacterial DNA, and their use for a routine testing scheme of Ea will be discussed.