Technical challenges for specific, sensitive detection of seed-borne bacterial pathogens

Abstract

Seed-borne pathogens are a major threat to agriculture production and security in a fast-moving global economy. As global trade increases so does the threat of accidental and deliberate introduction of seed-borne pathogens. Seed transmitted diseases can result in severe economic losses. The challenge is for government and industry to cooperate in providing pathogen-free seeds. Seeds can be assayed and infested lots destroyed or treated by chemical and/or physical means and re-assayed. Considerable progress has been made in developing reliable sensitive and specific assays. However, technical challenges remain. Seeds are challenging because they are often heavily contaminated with saprophytic bacteria. This makes agar plating difficult and often inhibits molecular-based protocols such as PCR. Use of DNA sequence information for designing highly specific and sensitive PCR primers is especially challenging. To avoid false negative results in classical PCR, internal controls of primers targeting bacterial 16S rDNA or plant 26S mitochondrial rDNA can be applied. Perhaps the most reliable and sensitive PCR protocol is real-time PCR using probe-based protocols such as TaqMan. The biggest challenge to PCR has been problems with PCR inhibitors present in seeds. These inhibitors can be partially removed using such treatments as heat, DNA extraction, immunocapture, and BIO-PCR. BIO-PCR not only reduces inhibitors but greatly increases sensitivity by allowing the target bacterium to multiply. This expands our ability to detect low numbers of pathogens even in seeds contaminated with large numbers of saprophytes. A major challenge for plant quarantine is dealing with PCR positive results without cultures for confirmation.