Clavibacter michiganensis subsp. michiganensis, Bacterial Canker of Tomato: 2. Comparison of the Effectiveness of Extraction Procedures and Sensitivity of Methods for Detection in Tomato Seeds

Abstract

Two seed extraction procedures, used for detection of Clavibacter michiganensis subsp. michiganensis (Cmm) in artifficially infested tomato seed lots, were evaluated. A comparison of the efficiency of pathogen detection by using different extraction methods showed that a grinding procedure was more effective than soaking seed samples. The extraction by grinding resulted in a higher number of samples with Cmm colonies than did the method that included soaking. The detection threshold of Cmm in relation to seed sample size was evaluated by adding different numbers of artificially infested seeds to uninfected samples of 2000 or 5000 seeds. Four detection methods were simultaneously compared for their sensitivity in Cmm detection in seeds: isolation on semiselective media (mSCM, D2ANX, mCNS), direct PCR from seed material, Bio-PCR with initial culturing of bacteria on NBY agar prior to PCR, and Enrichment PCR. The pathogen was detected in samples of 2000 seeds containing one, five and ten infested seeds, in at least two out of three replicates by three detection methods (selective plating, direct PCR and Bio-PCR), using the grinding extraction method with an addition of centrifugation step. In samples of 5000 seeds, five infested seeds were detected in all replicates by the same detection methods. Similar results were obtained by the soaking extraction method. In Enrichment PCR, positive results were obtained only in samples of 2000 seeds containing five and ten infested seeds regardless of the extraction method.

Ocenjivane su dve procedure ekstrakcije koje se primenjuju u detekciji Clavibacter michiganensis subsp. michiganensis (Cmm) u uslovima veštačke inokulacije uzoraka semena paradajza. Poređenje efikasnosti detekcije patogena korišćenjem metoda mlevenja i potapanja semena pokazalo je da je metod mlevenja bio efikasniji. Ekstrakcija mlevenjem semena rezultirala je neznatno veæim brojem uzoraka sa kolonijama Cmm nego procedura potapanja semena. Prag detekcije Cmm u zavisnosti od velièine uzorka ocenjivan je dodavanjem različitog broja veštački inokulisanih semena u nezaražene uzorke semena veličine 2000 i 5000 semena. Istovremeno je poređena osetljivost četiri metode za detekciju Cmm na semenu: izolacija na poluselektivne podloge (mSCM, D2ANX, mCNS), direktni PCR iz uzoraka semena, Bio-PCR koji uključuje gajenje bakterija na NBY podlozi pre PCR reakcije i Enrichment PCR. Patogen je detektovan u uzorcima veličine 2000 semena u koje su dodati jedno, pet i deset veštački zaraženih semena, u najmanje dva od tri ponavljanja korišćenjem tri metode za detekciju (izolacija na poluselektivne podloge, direktni PCR i Bio-PCR), nakon ekstrakcije mlevenjem uzoraka. Istim metodama, u uzorcima velièine 5000 semena, pet zaraženih semena je detektovano u svim ponavljanjima. Slični rezultati su dobijeni i nakon ekstrakcije potapanjem semena. U Enrichment PCR reakciji, pozitivni rezultati su dobijeni samo u uzorcima veličine 2000 semena u koje je dodato pet i deset zaraženih semena nezavisno od toga koja je procedura ekstrakcije korišćena.