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

Authors: MOEIN, S - University Of Stellenbosch; Mazzola, Mark; SPIES, C - Agricultural Research Council Of South Africa; MCLEOD, A - University Of Stellenbosch

Submitted to: European Journal of Plant Pathology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 12/4/2018
Publication Date: 1/3/2019
Citation: Moein, S., Mazzola, M., Spies, C.F., Mcleod, A. 2019. Evaluating different approaches for the quantification of oomycete apple replant pathogens, and their relationship with seedling growth reductions. European Journal of Plant Pathology. 154:243-257.

Interpretive Summary: Replant disease of apple is a major limiting factor to the development of an economically viable orchard on sites previously planted to this crop. A major constraint to development of environmentally sustainable disease control methods is a lack of knowledge as to the importance of specific root pathogens to overall symptoms expressed by apple trees in the orchard. These studies were conducted to determine whether a DNA-based protocol would be more precise than a traditional cultivation-based method to correlate the presence of a particular pathogen and the resulting inhibition of tree growth observed in the field. The study focused on several pathogens in a group of microorganisms termed oomycetes (including Pythium sylvaticum, Pythium irregulare, Pythium ultimum, P. vexans and Phytophthora cactorum), all of which contribute to the development of apple replant disease in South Africa and the United States. The cultivation-based method of determining plant root pathogen infection was correlated with relative growth reduction of apple growth incited by all of the pathogens with the exception of Phytophthora cactorum. The DNA-based protocol resulted in a negative correlation between pathogen DNA quantity and apple growth suppression for Pythium sylvaticum, Pythium irregulare, Pythium ultimum, but not P. irregulare and Phytophthora cactorum. These findings demonstrate that quantification of P. cactorum root infection by either method is not likely to accurately depict the relative contribution of this pathogen to overall growth suppression. However, the DNA protocol appears to be useful in determining overall infestation of planting stock from the nursery and, correspondingly, subsequent impaired tree growth, incited by P. sylvaticum, P. vexans and P. ultimum.

Technical Abstract: Investigations into inoculum sources and disease management strategies require effective pathogen quantification techniques, which should ideally be reflective of the extent of plant damage. The current study investigated whether determination of relative pathogen DNA quantity in root tissue improves 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 E. 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) from the glasshouse trials were negatively correlated with seedling growth reductions incited by P. sylvaticum, P. vexans and P. ultimum, when the fine feeder root systems of seedlings from the glasshouse trial were targeted. This, however, was not true for P. cactorum and P. irregulare. The percent infected roots also had a significant negative correlation with seedling growth reductions for P. sylvaticum, P. vexans, P. ultimum and P. irregulare, but not for P. cactorum.