A multiplex PCR method for detection of Clavibacter michiganensis subsp. michiganensis with co-amplification of its host DNA

Abstract

A multiplex PCR assay system was developed for the detection of Clavibacter michiganensis subsp. michiganensis (Cmm), which combined two tests in one reaction mixture. Cmm-specific primers PSA-4/PSA-R and Solanum lycopersicum–specific primers NS-7-F/NS-8-R (internal PCR control primer) were combined in one PCR reaction mixture with Cmm and plant DNA as template. The primer sets could amplify the target product successfully. Different combinations and concentrations of primers and annealing temperatures were tested, respectively. The detection level of the optimized multiplex PCR assay was up to 5×10² cfu·mL^-1. To verify the applicability of this system, it was employed to detect Cmm in tomato seeds and plantlet samples. Seeds mixed with Cmm and diseased plantlets were detected successfully. The multiplex PCR system will avoid false-negative results and provide a reliable method for the detection of Cmm.

Key words： Clavibacter michiganensis subsp. michiganensis (Cmm); molecular detection; multiplex PCR; internal PCR control; false negative

Acknowledgements

This research was financially supported by the doctor’s foundation of Hebei Province, China (No. 05547005D-1).

References

1 Burger A, Gräfen I, Engemann J, Niermann E, Pieper M, Kirchner O, Gartemann K H, Eichenlaub R (2005). Identification of homologues to the pathogenicity factor Pat-1, a putative serine protease of Clavibacter michiganensis subsp. michiganensis. Microbiological Research, 160: 417-427

2 Burokienė D (2006). Early detection of Clavibacter michiganensis subsp. michiganensis in tomato seedlings. Agronomy Research, 4(Suppl): 151-154

3 Chamberian J S, Gibbs R A, Ranier J E, Nguyen P N, Thomas C (1988). Detection screening of the duchenne muscular dystrophy locus via multiplex DNA amplification. Nucleic Acids Research, 16: 1141-1156

4 Dreier J, Bermpohl A, Eichenlaub R (1995). Southern hybridization and PCR for specific detection on phytopathogenic Clavibacter michiganensis subsp. michiganensis. Phytopathology, 85: 462-468

5 Elnifro E M, Ashshi A M, Cooper R J, Klapper P E (2000). Multiplex PCR: optimization and application in diagnostic virology. American Society for Microbiology, 13(4): 559-570

6 EPPO/CABI (2005). Clavibacter michiganensis subsp. michiganensis. EPPO Bull, 35: 275-283

7 Fu P, Guo Y H, Zhang X M, Guo J H (2005). Development of a PCR assay for the detection of Clavibacter michiganensis subsp. michiganensis. Jiangsu Journal of Agricultural Sciences, 21(2): 118-122 (in Chinese)

8 Gitaitis R, McCarter S, Jones J (1992). Disease control in tomato transplants produced in Georgia and Florida. Plant Disease, 76: 651-656

9 Janse J D, Wenneker M (2002). Possibilities of avoidance and control of bacterial plant diseases when using pathogen-tested (certified) or treated planting material. Plant Pathology, 51: 523-536

10 Luo L X, Li J Q, Bolka H (2005). Study on a new inoculating method for bacterial canker of tomato seedlings caused by Clavibacter michiganensis subsp. michiganensis. Acta Phytopathologica Sinica, 35(2): 123-128 (in Chinese)

11 Luo L X, Zhao Y C, Li J Q, Zhang L, Li Y (2000). Progress in research on bacterial canker of tomato caused by Clavibacter michiganensis subsp. michiganensis. Scientia Agriculture Sinica, 37(8): 1144-1150 (in Chinese)

12 Nordstrom J L, Vickery M C L, Blackstone G M, Murray S L, DePaola A (2007). Development of a multiplex real-time PCR assay with an internal amplification control for the detection of total and pathogenic vibrio parahaemolyticus bacteria in Oysters. American Society for Microbiology, 73(18): 5840-5847

13 Pastrik K H (2000). Detection of Clavibacter michiganensis subsp. sepedonicus in potato tubers by multiplex PCR with coamplification of host DNA. European Journal of Plant Pathology, 106: 155-165

14 Pastrik K H, Rainey F A (1999). Identification and differentiation of Clavibacter michiganensis subspecies by polymerase chain reaction-based techniques. Journal of Phytopathology, 147: 687-693

15 Sousa Santos S, Cruz L, Norskov P, Rasmussen O F (1997). A rapid and sensitive detection of Clavibacter michiganensis subsp. michiganensis in tomato seeds by polymerase chain. Seed Sci & Technol, 25: 581-584

16 White T J, Bruns T, Lee S, Taylor J W (1990). Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: Innis M A, Gelfand D H, Sninsky J J, White T J, eds. PCR Protocols: A Guide to Methods and Applications. New York and London: Academic Press, Incorporated, 315-322

17 Zhang L S, Becquet V, Li S H, Zhang D (2003). Optimization of multiplex PCR and multiplex gel electrophoresis in sunflower SSR analysis using infrared fluorescence and tailed primers. Acta Botanica Sinica, 45: 1312-1318

18 Zhao W J, Xia M X, Chen H Y, Zhu S F, Tan T W (2007). Rapid detection of Clavibacter michiganensis subsp. michiganensis using PCR. Plant Quarantine, 21(2): 75-77 (in Chinese)