Grafting connects root and shoot systems of distinct individuals, bringing microbial communities of different genotypes together in a single plant. How do root system and shoot system genotypes influence plant microbiota in grafted grapevines? To address this, we utilized clonal replicates of the grapevine ‘Chambourcin’, growing ungrafted and grafted to three different rootstocks in three irrigation treatments. Our objectives were to (1) characterize the microbiota (bacteria and fungi) of below-ground compartments (roots, adjacent soil) and above-ground compartments (leaves, berries), (2) determine how rootstock genotype, irrigation, and their interaction influences grapevine microbiota in different compartments, and (3) investigate abundance of microorganisms implicated in the late-season grapevine disease sour rot (Acetobacterales and Saccharomycetes). We found that plant compartment had the largest influence on microbial diversity. Neither rootstock genotype nor irrigation significantly influenced microbial diversity or composition. However, differential abundance of bacterial and fungal taxa varied as a function of rootstock and irrigation treatment; in particular, Acetobacterales and Saccharomycetes displayed higher relative abundance in berries of grapevines grafted to ‘1103P’ and ‘SO4’ rootstocks and varied across irrigation treatments. This study demonstrates that grapevine compartments retain distinct microbiota and identifies associations between rootstock genotypes, irrigation treatment, and the relative abundance of agriculturally relevant microorganisms in the berries.

Sour rot, a disease affecting berries of cultivated Vitis spp. worldwide, has not been clearly defined. Reported symptoms of the disease include browning of the berry skin, oozing of disintegrated berry pulp, and the smell of acetic acid, all in the presence of fruit flies (Drosophila spp.). We determined acetic acid concentrations in multiple collections of symptomatic berries, isolated and identified microbes from them, and inoculated commonly isolated organisms into healthy berries with and without concurrent exposure to wild-type or axenic Drosophila melanogaster. Coinoculations combining one of several yeasts (Metschnikowia spp., Pichia spp., and a Saccharomyces sp.) plus an acetic acid bacterium (an Acetobacter sp. and Gluconobacter spp.) reproduced sour rot symptoms, defined here as decaying berries with a loss of turgor and containing acetic acid at a minimum of 0.83 g/liter, based on observed field levels. Symptoms developed only in the presence of D. melanogaster, either wild type or axenic, indicating a nonmicrobial contribution of these insects in addition to a previously suggested microbial role. We conclude that sour rot is the culmination of coinfection by various yeasts, which convert grape sugars to ethanol, and bacteria that oxidize the ethanol to acetic acid, and that this process is mediated by Drosophila spp.

Sour rot is a disease complex characterized by rotting of the grape berry plus internal development of acetic acid, typically associated with an abundance of Drosophila fruit flies. Uncertainties regarding disease etiology and epidemiology have limited the development of reliable management practices. It is now known that yeast, acetic acid bacteria (AAB) and Drosophila spp. act together to cause the disease. Thus, we conducted three years of replicated field trials on the Vitis interspecific hybrid cv. Vignoles, in which we targeted these organisms through pre-harvest applications of various antimicrobial agents (potassium metabisulfite, copper hydroxide, BLAD polypeptide, and/or a mixture of hydrogen dioxide and peroxyacetic acid, depending on year) and an insecticide (spinetoram or zeta-cypermethrin, depending on year), both alone and in combination. Weekly applications of an antimicrobial plus insecticide provided an average 64% control relative to untreated vines across all three years of the trial when initiated preventively at 15 Brix, before the onset of symptoms; withholding addition of an antimicrobial to the insecticide application until symptoms appeared typically decreased the control level. Applying only an insecticide on the preventive schedule provided substantial control in two of the three years, and significantly reduced the number of drosophilids recovered from the berries within the treated panels, whereas the antimicrobials were ineffective unless also applied with insecticide. Additionally, we studied disease development in a commercial vineyard of cv. Vignoles in which vines are trained to either a high wire cordon (HW) or vertical shoot position (VSP) system in groups of adjacent rows. In all three years of monitoring, disease severity was significantly higher on vines in the HW system where drooping shoots enclosed fruit within an umbrella-like canopy, whose density between the fruiting zone and vineyard floor was greater than for VSP vines.

Sour rot is a disease complex produced by an interaction between grape berries and various species of yeast and acetic acid bacteria in the presence of Drosophila fruit flies. While yeast and bacteria are consistently found on healthy grape berries worldwide, we explored whether the composition of these epiphytic communities differed depending on the presence or absence of sour rot symptoms. Using high-throughput sequencing, we characterized the microbiome of sour rot-affected grapes from two geographical areas across two years. In 2015 and 2016, both healthy and sour rot-affected berries were collected from commercial and research vineyards in Geneva, NY and commercial vineyards in Tasmania, AUS. In this experiment, all associated organisms grouped together primarily by location, and not by presence/absence of symptoms or cultivar. The predominant difference between asymptomatic and symptomatic samples, regardless of location, was the abundance of Acetobacter species, which were significantly more plentiful in the symptomatic samples. Yeast genera such as Candida, Hanseniaspora, Pichia and Saccharomyces were abundant in both sets of samples, but varied by region. The consistent presence of yeast species and the increased abundance of acetic acid-generating bacteria is consistent with our understanding of their etiological role in sour rot development. In 2016, diseased grapes also were collected from vineyards in Fredonia, NY, and Modesto, CA. Consistent with our comparison study, all associated organisms again grouped together primarily by location. Yeast genera such as Candida, Hanseniaspora, Pichia and Saccharomyces were abundant in both sets of samples, but varied by region. The consistent presence of yeast species and the abundance of acetic acid-generating bacteria in both experiments is consistent with our understanding of their etiological role in sour rot development.

Intact, healthy grape berries were sampled from vineyards in the states of Washington and New York, USA and Tasmania, Australia, and from bunches of table grapes exported from Chile that were purchased on two occasions in a U.S. supermarket. Endophytic microbes were isolated on media conducive to fungi or bacteria and subsequently identified by Illumina sequencing of their DNA. Species of the yeast genera Metschnikowia, Pichia, and Hanseniaspora were recovered from every set of samples, as were species of the bacterial genera Acinetobacter, Burkholderia and Bacillus; species of the bacterial genera Acetobacter and Gluconobacter also were recovered from vineyard samples from New York and Tasmania and from supermarket-purchased grapes. Multiple other fungal and bacterial species were recovered less often. When quantified for the Washington samples and one set from the supermarket, non-Saccharomyces yeast species represented the vast majority of fungal identifications, whereas the distribution of various bacterial species varied widely between and within the two sources. The endophytic presence of these microbes within grape berries has implications with respect not only to the potential development of sour rot but also to the broader concept of microbial terroir in wine quality.

Isolating DNA from microbes on the surface of a grape berry is a challenge due to their adhesion to the thick berry skin and cuticle, making studies of the grape microbiome challenging. We developed a field-to-lab DNA extraction procedure that starts in the vineyard, disrupts the grape berry surface while en route to the lab through agitation, and efficiently extracts microbial DNA from the surface of the grape. It is cost effective and utilizes commonly available laboratory chemicals with low toxicity (Table 1). This protocol allows researchers to extract DNA from the grape berry surface in the field, therefore undergoing minimal manipulation of those microbiomes before DNA extraction.