Winemakers have long used copper as a conventional fungicide treatment on grapevine and only recently, the use of biocontrol agents depicted a promising alternative. Most of the studies that investigate the impact of fungicide treatments, focus on specific pathogenic microbes. In the present work instead, a field experiment conducted in South Africa shows the seasonal microbial change occurring on grapevine leaves, periodically treated with two different fungicide treatments: copper sulphate and Lactobacillus plantarum MW-1. In this work, NGS data were combined with strain-specific and community qpcRs to reveal the shift of the microbial communities throughout the growing season and highlight the impact of fungicides on the microbiota. Only the family of Lactobacillaceae systematically changed between treatments, while the bacterial community remained relatively stable over time. MW-1 was exclusively detected on biocontrol-sprayed leaves. Conversely, the fungal community was largely shaped by sampling time, underlining the succession of different dominant taxa over the months. Between treatments, only a few fungal taxa appeared to change significantly and the fungal load was also comparable. Monitoring the dynamics of the microbial community under different fungicide treatments may advise the best timing to apply treatments to the plants, toward the realization of more sustainable agricultural practices.

Soil DNA extraction encounters numerous challenges that can affect both yield and purity of the recovered DNA. Clay particles lead to reduced DNA extraction efficiency, and PCR inhibitors from the soil matrix can negatively affect downstream analyses when applying DNA sequencing. Further, these effects impede molecular analysis of bacterial community compositions in lower biomass samples, as often observed in deeper soil layers. Many studies avoid these complications by using indirect DNA extraction with prior separation of the cells from the matrix, but such methods introduce other biases that influence the resulting microbial community composition. To address these issues, a direct DNA extraction method was applied in combination with the use of a commercial product, the G2 DNA/RNA Enhancer, marketed as being capable of improving the amount of DNA recovered after the lysis step. The results showed that application of G2 increased DNA yields from the studied clayey soils from layers from 1.00 to 2.20 m. Importantly, the use of G2 did not introduce bias, as it did not result in any significant differences in the biodiversity of the bacterial community measured in terms of alpha and beta diversity and taxonomical composition. Finally, this study considered a set of customised lysing tubes for evaluating possible influences on the DNA yield. Tubes customization included different bead sizes and amounts, along with lysing tubes coming from two suppliers. Results showed that the lysing tubes with mixed beads allowed greater DNA recovery compared to the use of either 0.1 or 1.4 mm beads, irrespective of the tube supplier. These outcomes may help to improve commercial products in DNA/RNA extraction kits, besides raising awareness about the optimal choice of additives, offering opportunities for acquiring a better understanding of topics such as vertical microbial characterisation and environmental DNA recovery in low biomass samples.

Soil DNA extraction encounters numerous challenges that can affect both yield and purity of the recovered DNA. Clay particles lead to reduced DNA extraction efficiency, and PCR inhibitors from the soil matrix can negatively affect downstream analyses when applying DNA sequencing. Further, these effects impede molecular analysis of bacterial community compositions in lower biomass samples, as often observed in deeper soil layers. Many studies avoid these complications by using indirect DNA extraction with prior separation of the cells from the matrix, but such methods introduce other biases that influence the resulting microbial community composition.To address these issues, a direct DNA extraction method was applied in combination with the use of a commercial product, the G2 DNA/RNA Enhancer®, marketed as being capable of improving the amount of DNA recovered after the lysis step. The results showed that application of G2 increased DNA yields from the studied clayey soils from layers between 1.00 and 2.20 m below ground level.Importantly, the use of G2 did not introduce bias, as it did not result in any significant differences in the biodiversity of the bacterial community measured in terms of alpha and beta diversity and taxonomical composition.Finally, this study considered a set of customised lysing tubes for evaluating possible influences on the DNA yield. Tubes customization included different bead sizes and amounts, along with lysing tubes coming from two suppliers. Results showed that the lysing tubes with mixed beads allowed greater DNA recovery compared to the use of either 0.1 or 1.4 mm beads, irrespective of the tube supplier.These outcomes may help to improve commercial products in DNA/RNA extraction kits, besides raising awareness about the optimal choice of additives, offering opportunities for acquiring a better understanding of topics such as vertical microbial characterisation and environmental DNA recovery in low biomass samples.

11Winemakers have long used copper as a fungicide on grapevine. However, the potential of copper to 12 accumulate on soil and affect the biota poses a challenge to achieving sustainable agriculture. One 13 recently developed option is the use of biocontrol agents to replace or complement traditional methods. 14 In the present study, a field experiment was conducted in South Africa in which the leaves in two blocks 15 of a vineyard were periodically treated with either copper sulphate or sprayed with Lactobacillus 16 plantarum MW-1 as a biocontrol agent. This study evaluated the impact of the two treatments on the 17 bacterial and fungal communities as they changed during the growing season. To do this, NGS was 18 combined with quantitative strain-specific and community qPCRs. The results revealed the progression 19 of the microbial communities throughout the season and how the different treatments affected the 20 microbiota. Bacteria appeared to be relatively stable at the different time points, with the only taxa that 21 systematically changed between treatments being Lactobacillaceae, which included reads from the 22 biocontrol agent. Cells of Lactobacillus plantarum MW-1 were only detected on treated leaves using 23 strain-specific qPCR, with its amount spanning from 10 3 to 10 5 cells/leaves. Conversely the fungal 24 community was largely shaped by a succession of different dominant taxa over the months. Between 25