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.

Wine grape production is an important economic asset in many nations; however, a significant proportion of vines succumb to grapevine trunk pathogens, reducing yields and causing economic losses. Cover crops, plants that are grown in addition to main crops in order to maintain and enhance soil composition, may also serve as a line of defense against these fungal pathogens by producing volatile root exudates and/or harboring suppressive microbes. We tested whether cover crop diversity reduced disease symptoms and pathogen abundance. In two greenhouse experiments, we inoculated soil with a 10⁶ conidia suspension of Ilyonectria liriodendri, a pathogenic fungus, then conditioned soil with cover crops for several months to investigate changes in pathogen abundance and fungal communities. After removal of cover crops, Chardonnay cuttings were grown in the same soil to assess disease symptoms. When grown alone, white mustard was the only cover crop associated with reductions in necrotic root damage and abundance of Ilyonectria. The suppressive effects of white mustard largely disappeared when paired with other cover crops. In this study, plant identity was more important than diversity when controlling for fungal pathogens in vineyards. This research aligns with other literature describing the suppressive potential of white mustard in vineyards.

(1) Background: Arbuscular mycorrhizal (AM) fungi are symbiotic organisms that help plants acquire nutrients from the soil in exchange for photosynthetic carbon. Commercial AM fungal inoculants are widely available and are used extensively in agriculture including wine grape production. However, positive growth responses from inoculants are more consistent in the greenhouse compared to the field. (2) Methods: We grew three grapevine rootstocks with and without an AM fungal inoculant in the greenhouse for one year, then they were transplanted to the field for two years. To quantify the establishment of the inoculant, we analyzed root samples with a digital PCR assay. (3) Results: We show that AM fungal inoculation increased biomass production only in the greenhouse. After two growing seasons in the field, the commercial inoculant colonized roots but did not increase biomass production compared to uninoculated rootstocks. (4) Conclusions: This study highlights that AM fungal inoculants do not always promote growth of grapevines in the field. Future research should focus on inoculant strains designed for viticulture applications and take rootstock into consideration to maximize their efficacy.