PCR detection ofclavibacter michiganensis subsp.sepedonicus- infected tuber samples in a plate capture assay

The speed and sensitivity of PCR-based assays allow shorter turnaround times for the detection of pathogens for which culture and serological methods are difficult or unavailable. PCR was performed with primer sets Cms50 and Cms72, designed previously by Millset al. (1997) through subtractive hybridization to detectClavibacter michiganensis subspeciessepedonicus (Cms). In bacterial suspensions, fewer than three cells/10 ul reaction were detected after PCR amplicons were hybridized with specific DIG-labeled DNA probes in an enzyme-linked oligonucleosorbent assay (ELOSA). In naturally infected tuber samples representing three cultivars of potato, the diagnostic sensitivity of PCR/ ELOSA was 96%, while the specificity exceeded 99%. PCR/ELOSA detectedCms in infected tuber samples with equal sensitivity regardless of colony morphology, potato cultivar, or primer sets.     

Extraction and Activation of Wheat Polyphenol Oxidase by Detergents: Biochemistry and Applications

Polyphenol oxidases (PPOs) from several plant species, including wheat, have been implicated in undesirable brown discolorations of food products. It has been demonstrated that these enzymes are often present in a latent form or are membrane‐associated, necessitating detergent or other treatments to obtain fully active preparations. Here, the influence of different detergents on wheat meal and flour PPOs was investigated. Extraction in presence of 50 mM SDS led to a 5‐ to 15‐fold increase in PPO activity, making quantitative assays in flour from low‐PPO lines more robust. Among a series of additional nonionic, anionic, and cationic detergents tested, only n ‐lauroylsarcosine increased extractable PPO activity to a degree comparable to that of SDS. Additional experiments suggested that a large fraction of wheat meal PPOs may be membrane‐associated and that SDS is able to activate PPOs extracted from high‐activity but not from low‐activity wheat lines. PPO activities assayed after SDS extraction of meal and flour were highly correlated with each other and with activity determined in whole (intact) kernels in absence of SDS. Correlation coefficients between PPO activities measured with all these methods and noodle brightness were about equal, indicating that activities assayed after SDS extraction are useful for germplasm screening and quality prediction.