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ARS Home » Northeast Area » Frederick, Maryland » Foreign Disease-Weed Science Research » Research » Publications at this Location » Publication #166853
Title: REAL-TIME PCR ASSAYS FOR DETECTING BACTERIA IN SEED AND OTHER PLANTING MATERIAL

Author
item Schaad, Norman
item BOLKAN, H - CAMPBELL SOUP RESEARCH
item RANDHAWA, P - CALIFORNIA PLANT & SEED

Submitted to: International Plant Protection Congress
Publication Type: Abstract Only
Publication Acceptance Date: 3/25/2004
Publication Date: 5/11/2004
Citation: International Plant Protection Congress Abstracts XV:63

Interpretive Summary:

Technical Abstract: A prerequisite to controlling any plant disease is rapid, reliable identification of the causal organism. The best control for seedborne bacteria is a reliable seed health testing program. Serological, isolation, and PCR assays have been described for several pathogens. However, these assays either lack sensitivity or specificity, or are very time consuming. A real-time PCR assay using fluorescent-labeled probes (TaqMan) is more robust and rapid due to a built-in probe. When combined with rapid cycling portable PCR platforms, and direct PCR, assays can be done in as little as one hour. Such PCR assays are actually less sensitive than agar plating assays and are often inhibited by plant extracts. However, by pre-enriching the extracted bacteria on agar media (termed BIO-PCR), inhibitors are eliminated and sensitivity increased 10-100-fold over standard PCR. Using a real-time TaqMan BIO-PCR assay and 30 asymptomatic tubers from ring rot suspect plants, the slow-growing Clavibacter michiganensis subsp. sepedonicus was detected in 28 tubers, whereas agar plating and standard real-time TaqMan PCR detected only 4 and 8 tubers, respectively. A real-time BIO-PCR assay for Ralstonia solanacearum (RS) bv2, using potato extracts spiked with RS, detected as few as 30 cells of RS/ml, whereas the standard real-time PCR assay needed 3x 10**3 cells/ml. Similarly, seedborne bacteria can be enriched in host seedlings or on Ampli-Discs prior to PCR. These techniques are much more effective than PCR performed directly on seed extracts in detecting Acidovorax avenae subsp citrulli and C. michiganensis subsp. michiganensis in watermelon and tomato seeds, respectively.