Detection of Clavibacter michiganensis subsp. sepedonicus in Potato Tubers by BIO-PCR and an Automated Real-Time Fluorescence Detection System

Abstract

Ring rot of potato, caused by Clavibacter michiganensis subsp. sepedonicus, is one of the most regulated diseases of potatoes world wide. The organism is often difficult to detect in symptomless tubers because of low populations and slow competitive growth on available media. Polymerase chain reaction (PCR) primers and a fluorescent probe for use in the Perkin Elmer 7700 automated real time PCR detection system (TaqMan) were designed from a C. michiganensis subsp. sepedonicus-specific genomic DNA fragment for development of a BIO-PCR assay for C. michiganensis subsp. sepedonicus in potato tubers. Results of screening the primers with strains of C. michiganensis subsp. sepedonicus and other bacteria showed the primers to be specific. A total of 30 naturally infected ring rot suspect tubers were sampled by the core extract, shaker incubation procedure and assayed by (i) plating aliquots onto agar media, (ii) classical PCR, and (iii) BIO-PCR. In all, 4 tubers were positive by agar plating and pathogenicity tests, 8 by classical TaqMan PCR, and 26 by TaqMan BIO-PCR. We conclude that BIO-PCR combined with the TaqMan automated closed detection system is a rapid, reliable method of assaying large numbers of potato tuber extracts for C. michiganensis subsp. sepedonicus. Furthermore, for a large central laboratory running large numbers of PCR assays, the high-throughput TaqMan system can reduce costs per sample over the more labor-intensive classical PCR.

Management of plant health risks associated with processing of plant-based wastes: A review

“…The main risk to compost is from disposal of infected potato tubers. There is very little information on the inoculum level required to cause potato ring rot disease, but information accumulated on the inoculum levels required to infect aubergine plants in the bioassay test suggest that levels of <10 cfu may cause infection in plants when artificially inoculated (Lelliott and Sellar, 1976 (Pastrik, 2000;Schaad et al, 1999). The use of a pre-incubation on growth media has the additional advantage of confirming bacterial viability.…”

“…The EPPO diagnostic protocol (EPPO 2006) recommends the use of either MTNA medium (Jansing and Rudolph, 1998) or NCP88 medium (de la Cruz et al, 1992). Detection limits of Taqman BIOPCR (where bacteria are isolated onto media and then tested using Taqman real time PCR) have been quoted as 2-3 cells ml -1 (Schaad et al, 1999).…”

Otimização da técnica de PCR para a detecção de Xanthomonas axonopodis pv. phaseoli em sementes de feijão

“…flaccumfaciens (6), e em outras culturas, como a batata, em que foi possível detectar a presença de Clavibacter michiganensis subsp. sepedonicus (19).…”

“…Relatos na literatura (19) afirmam que a técnica de BIO-PCR possui muitas vantagens comparada aos métodos padrões para PCR, principalmente para a detecção de patógenos em sementes em análises de rotina. Dentre as vantagens, pode-se destacar a simplicidade da técnica, o aumento da sensibilidade e a detecção somente de células vivas.…”

Detection of Clavibacter michiganensis subsp. sepedonicus by AmpliDet RNA, a new technology based on real time monitoring of NASBA amplicons with a molecular beacon

“…Mills et al (1997) selected three primer pairs by genomic subtraction. On the basis of one of these pairs, a TaqMan PCR procedure was developed (Schaad et al 1999). Li et al (1997) developed a fluorescence in situ hybridization procedure that allowed double staining of cells with a DNA probe and antibodies.…”