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Droplet digital polymerase chain reaction (ddPCR) is a method used to detect and quantify nucleic acids even when present in exceptionally low numbers. While it has proven to be valuable for clinical studies, it has failed to be widely adopted for environmental studies but despite some limitations, ddPCR may represent a better option than classical qPCR for environmental samples. Due to the complexity of the chemical and biological composition of environmental samples, protocols tailored to clinical studies are not appropriate, and results are difficult to interpret. We used environmental DNA samples originating from field studies to determine a protocol for environmental samples. Samples included field soils which had been inoculated with the soil fungus Rhizophagus irregularis (environmental positive control), field soils that had not been inoculated and the targeted fungus was not naturally present (environmental negative control), and root samples from both field categories. To control for the effect of soil inhibitors, we also included DNA samples of an organismal control extracted from pure fungal spores (organismal positive control). Finally, we included a no-template control consisting only of the PCR reaction reagents and nuclease free water instead of template DNA. Using original data, we examined which factors contribute to poor resolution in root and soil samples and propose best practices to ensure accuracy and repeatability. Furthermore, we evaluated manual and automatic threshold determination methods and we propose a novel protocol based on multiple controls that is more appropriate for environmental samples.

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Ilyonectria is a weak pathogen known for causing black foot disease in young vines, infecting roots and vascular tissues at the basal end of the rootstock and restricting the movement of water and nutrients. This negatively impacts vine establishment during transplant into the vineyard. Arbuscular mycorrhizal (AM) fungi are symbiotic fungi that associate with most plants and have been shown to mitigate the infection and effect of pathogens. This greenhouse study was designed to determine if the mycorrhizal fungi could mitigate Ilyonectria infection and whether this was dependent on inoculation timing. ‘Riparia gloire’ grapevine rootstocks (Vitis riparia) were infected with Ilyonectria either after AM fungi, at the same time as AM fungi, or to roots that were not inoculated by AM fungi. We measured the abundance using specific markers for both the pathogen and AM fungi. Colonization by AM fungi did not suppress Ilyonectria, but instead increased the abundance of Ilyonectria. Further, mycorrhizal rootstocks did not have enhanced growth effects on physiological parameters when compared to non-mycorrhizal rootstocks. These findings stand in contrast to the general perception that AM fungi provide protection against root pathogens.

828 I. What is codependency and why should we care? 829 II. Should we expect codependency among AM communities? 830 III. Requirements for codependency 830 IV. When and where has codependency been observed? 831 V. Is codependency scale-and resolution-dependent? 831 VI. Unexplained variation: if not codependency then what? 834 VII Recommendations for future studies 834 VIII. Conclusion 835 Acknowledgements 835 References 835