Research On Molecular Detection Methods Of CPA For Clavibacter Michiganensis Plant Pathogens

All known subspecies of Clavibacter michiganensis (Cm) are subdivided into five subspecies:Clavibacter michiganensis subsp. michiganensis (Cmm) is the casual agent of bacterial wilt and canker of tomato; Clavibacter michiganensis subsp. sepedonicus (Cms) give rise to potato ring rot; Clavibacter michiganensis subsp. insidious (Cmi) causes wilting and stunting in alfalfa; Wilt and blight of maize is induced by Clavibacter michiganensis subsp. nebraskensis (Cmn) and Clavibacter michiganensis subsp. tessellarius (Cmt) causes leaf freckles and pots in wheat. Among the five bacteria, Clavibacter michiganensis subsp. michiganensis (Cmm)、Clavibacter michiganensis subsp. insidious (Cmi) and Clavibacter michiganensis subsp. nebraskensis are quarantine pests for our country. Therefore, it is very essential to develop rapid and sensitive diagnostic methods to detect these bacteria and limit the effects of diseases.In this paper, foused on the five pathogens of Clavibacter michiganensis, a simple, rapid, sensitive and specific detection system has been established based on CPA (Cross Priming Amplification) combined with gold-labeled DNA strip sensor. This diagnostic method does not need any expensive instruments and can be used to screen for bacterial plant disese.Three primers and two probes were developed based on the ITS sequences of Clavibacter michiganensis subspecies. This protocol was used to specially detect Clavibacter michiganensis plant pathogens and the results were compared and identified by routine PCR and real-time PCR respecitively. The assay showed that the CPA method can specifically detect the object strains from pure bacterial culture, the sensitivity of the five pathogens was as follow, Cmm:1.32×104cfu/mL; Cms:1.48×104cfu/mL; Cmi:4.75×103cfu/mL; Cmn:2.35×104cfu/mL; Cmt:1.78×103cfu/mL. The sentivity was almost the same as real-time PCR and higher than routine PCR.In the detection experiment of tomato seeds, as few as5.62×105cfu/mL can be successfully detected with sample DNA extraction from practically and artificially infected tomato seeds. The research established the detection method for Clavibacter michiganensis plant pathogens based on cross priming amplification (CPA) techonology. In the entire detection procedure, from sample processing to visual readout, all the instruments needed are heater and centrifuge. The new method created in this paper made the whole detection procedure more accurate, sensitive and rapid. It can effectively make up for the disadvanges of routine PCR and real-time PCR at the grassroots level. However, the method based on CPA in this research cannot distinguish the five subspecies; it has significant popularizing value and application prospect.