Evaluating different approaches for the quantification of oomycete apple replant pathogens, and their relationship with seedling growth reductions

Investigations into inoculum sources and disease management strategies require effective pathogen quantification techniques, which should ideally also be reflective of the extent of plant damage. The current study investigated whether determination of relative pathogen DNA quantity in root tissue can improve the assessment of plant damage by several oomycete apple replant pathogens when compared to absolute DNA quantifications and percent roots infected. Published real-time quantitative PCR (qPCR) assays were utilized to quantify pathogen DNA, except for Phytopythium vexans for which a new qPCR assay was developed. Relative pathogen DNA quantifications employed a mutated Escherichia coli gene spiked into the DNA extraction buffer. Pathogen quantifications were not improved through relative DNA quantifications since relative DNA quantities were highly and significantly correlated with absolute pathogen DNA quantities. This was evident from: (i) glasshouse experiments where five oomycete apple replant disease pathogens (Pythium sylvaticum, Pythium irregulare, Pythium ultimum, P. vexans and Phytophthora cactorum) were quantified from artificially inoculated apple seedlings roots, and (ii) quantification of P. irregulare from naturally-infected nursery tree roots. Relative- and absolute pathogen DNA quantities in infected glasshouse seedling roots (all five species) and nursery tree roots (P. irregulare), were furthermore significantly correlated with percent roots infected. Pathogen root DNA quantities (relative and absolute) obtained from the fine feeder root systems of seedlings from the glasshouse trials were significantly negatively correlated with increase in seedling length for P. sylvaticum, P. vexans and P. ultimum infected seedlings. This, however, was not true for P. cactorum and P. irregulare. The percent infected roots also had a significant negative correlation with increase in seedling length for P. sylvaticum, P. vexans and P. ultimum and P. irregulare, but not for P. cactorum.