Pathogen Enrichment And Rapid Molecular Biology Detection Methods For Tomato Bacterial Canker Pathogens, Clavibacter Michiganensis Subsp. Michiganensis

Objective:Tomato bacterial canker is the most important bacterial diseases which caused substantial economic losses in the worldwide. Seed is the main long-distance spread pathway. A small amount of bacteria in seed can lead to a large-scale prevalence, Seed born Clavibacter michiganensis subsp. michiganensis(Cmm) is difficult to detect by nomal methods because of its very low rate of infected-seed. Therefore, it is significance to prevent its invasion and spread by establishing an accurate, rapid and sensitive method for Cmm detection.Methods:Comparison of several methods for the detection of Cmm in bacterial suspensions and experimentally contaminated seed samples. These methods including immunological method such as ELISA, molecular biology methods such as Direct-PCR and Nested-PCR, as well as the methods of pathogen enrichment combined with PCR such as BIO-PCR and nitrocellulose membrane capture PCR, the methods of pathogen concentration combined with PCR such as membrane filter PCR, immunocapture PCR(IC-PCR) and immunomagnetic separation PCR(IMS-PCR). Then, We evaluate the sensitivity, testing period, stability and the cost of these methods.Results:The immunological method and molecular biology methods such as ELISA, Direct-PCR and Nested-PCR were performed in bacterial suspensions and experimentally contaminated seed samples. These results were compared with the methods of pathogen enrichment or concentration combined with PCR such as nitrocellulose membrane capture PCR, BIO-PCR, membrane filter PCR, IC-PCR and IMS-PCR. ELISA detection limit is more than 106 cfu/mL of pathogen in all assayed samples. The detection sensitivity of Direct-PCR is 104 cfu/mL and 106 cfu/mL in bacterial suspensions and experimentally contaminated seed samples, respectively. Nested-PCR arrived 102 cfu/mL and 104 cfu/mL in bacterial suspensions and experimentally contaminated seed samples, respectively. BIO-PCR provided the positive results for 10 cfu/mL in bacterial suspensions and experimentally contaminated seed samples. IC-PCR arrive 102 cfu/mL and 104 cfu/mL in bacterial suspensions and experimentally contaminated seed samples, respectively. The detection limite of IMS-PCR is 102 cfu/mL in two assayed samples.Conclusion:The methods of immunology technique combined with molecular biology techniques such as IC-PCR and IMS-PCR provided the best results regarding sensitivity and specificity for the detection of seed born Cmm, which are deserved to extend and exertion.