Silvia Alejandra

Villarreal Soto

Jalloul Bouajila

Mauro Pace

John Leech

Paul D Cotter

Jean-Pierre Souchard

Patricia Taillandier

Sandra Beaufort
email: sandra.beaufort@ensiacet.fr

Silvia Alejandra Villarreal-Soto

Metabolome-microbiome signatures in the fermented beverage, Kombucha

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Successful interactions between bacteria and yeast species can lead to the generation of a wide range of metabolites with interesting bioactivities, including those resulting from fermented beverage production. The microbial community responsible for kombucha (fermented tea) production is embedded in an extracellular polymeric matrix (pellicle) that is located at the liquid-air interphase [START_REF] Nikolaev | Biofilm -"City of microbes" or an analogue of multicellular organisms[END_REF]. The tea is also commonly inoculated with 10-20% of broth from a previous kombucha fermentation ( [START_REF] Četojević-Simin | Bioactivity of lemon balm Kombucha[END_REF][START_REF] Malbaša | Scale-up the products of kombucha fermentation on molasses[END_REF][START_REF] Rosma | Fermentation of black tea broth (Kombucha): I. Effects of sucrose concentration and fermentation time on the yield of microbial[END_REF], allowing the microbial population to disperse in the liquid media. The characteristic kombucha microbiome includes several genera of acetic acid bacteria, yeasts and, to a lesser extent, lactic acid bacteria. Some studies [START_REF] Coton | Unraveling microbial ecology of industrial-scale kombucha fermentations by metabarcoding and culture-based methods[END_REF][START_REF] Marsh | Sequence-based analysis of the bacterial and fungal compositions of multiple kombucha (tea fungus) samples[END_REF] have revealed Komagataeibacter, Acetobacter and Gluconobacter as the dominant bacteria genera, and Brettanomyces and Zygosaccahromyces as the dominant yeast. These microorganisms interact through cooperative metabolism, contributing to the synthesis and organoleptic qualities of the final beverage. Several metabolic reactions occur during kombucha fermentation leading to the production of intermediates and secondary metabolites, with potential applications in the pharmaceutical, cosmetic and food industries (Villarreal-Soto et al., 2018). Acetic acid bacteria are known to convert sugars and alcohols into several organic acids, such as acetic, gallic, succinic and malic acids [START_REF] Gomes | Acetic Acid Bacteria in the Food Industry : Systematics[END_REF]. The genus Gluconacetobacter and Komagataeibacter are cellulose producing bacteria that also participate in the production of gluconic and glucuronic acid by an oxidation reaction with the enzyme glucose dehydrogenase [START_REF] Ramachandran | Gluconic acid : properties, applications and microbial production[END_REF]. Yeasts are also important contributors due to their high fermentative capabilities, releasing high quantities of polysaccharides, great efficiency with respect to the utilization of available nitrogen sources and high level of resistance to osmotic and ethanol stress [START_REF] Domizio | Cell wall polysaccharides released during the alcoholic fermentation by Schizosaccharomyces pombe and S. japonicus: quantification and characterization[END_REF][START_REF] Steensels | Brettanomyces yeasts -from spoilage organisms to valuable contributors to industrial fermentations[END_REF]. Thus, the production of important compounds is directly related to the specific microbiota and the cross-feeding between them. In addition to the composition of the pellicle, the factors that will dictate the growth and activity of kombucha consortia include the substrate [START_REF] Ayed | Development of a beverage from red grape juice fermented with the kombucha consortium[END_REF][START_REF] Četojević-Simin | Bioactivity of lemon balm Kombucha[END_REF][START_REF] Hoon | Kombucha "tea fungus" enhances the tea polyphenol contents, antioxidant activity and alpha-amylase inhibitory activity of five commonly consumed teas[END_REF][START_REF] Rahmani | Kombucha fermentation of African mustard (Brassica tournefortii) leaves : chemical composition and bioactivity[END_REF] T resuspended in 550 μL of SL buffer. The sample was then treated following the same protocol mentioned above.

Kombucha samples (liquid and solid phases from the end of the fermentation) were sequenced on the NextSeq sequencing platform Illumina, (San Diego, CA, USA) in the Teagasc sequencing facility (Moorepark, Cork, Ireland). Library preparation was carried out according to the Illumina Nextera XT protocol Illumina Inc., (San Diego, CA, USA). Quantification of DNA was performed using the Invitrogen Qubit High Sensitivity dsDNA assay (ThermoFisher scientific, Waltham, MA, USA). Final library quality was assessed by running on an Agilent High Sensitivity DNA chip (Agilent Technologies, Waldbroon, Germany), and quantification by qPCR using the KAPA Library Quantification Kit for Illumina (Roche). Sequencing was carried out using a 300 cycle High Output V2 kit, following standard Illumina sequencing protocols as described by [START_REF] Doyle | High-throughput metataxonomic characterization of the raw milk microbiota identi fi es changes reflecting lactation stage and storage conditions[END_REF].

Sequence reads were obtained from the Nextseq sequencing run in the form of Bcl files. These files were converted to fastq format using bcl2fastq v. 2.19 illumina software. Using Picard v. 2.7.1 (available at http://picard.sourceforge.net), fastq was converted to Sam format. Picard was also used to remove duplicates. The sequences were then quality checked and trimmed using bwa v. 0.7.15 [START_REF] Li | Fast and accurate short read alignment with Burrows-Wheeler transform[END_REF]. Forward and reverse reads were combined into a single fasta file for each sample.

Kaiju [START_REF] Menzel | Fast and sensitive taxonomic classification for metagenomics with Kaiju[END_REF] was used to assign taxonomy to the reads, discarding taxa with relative abundance of less than 0.1%. This setting was chosen as other studies have shown a high false positive discovery rate below this threshold. All percentages reported at all taxonomic levels are percentages of the assigned reads only. Summary plots were created in R version 3.6.0 (R Core Team, 2014) using the package ggplot2 v. 3.1.0 [START_REF] Wickham | Ggplot2: Elegant Graphics for Data Analysis, 2nd Edition[END_REF]. Superfocus v. 0.34 [START_REF] Silva | Sequence analysis SUPER-FOCUS : a tool for agile functional analysis of shotgun metagenomic data[END_REF] and was used to assign functionality to the reads.

Melonnpan v. 0.99 [START_REF] Mallick | Predictive metabolomic profiling of microbial communities using amplicon or metagenomic sequences[END_REF] (Predictive metabolomic profiling of microbial communities using amplicon or metagenomic sequences) was used to predict the metabolites produced from each microbiome. The top 50 predicted metabolites were then plotted using ggplot2 v. 3.1.0 in0 R.

Metagenome assembly was carried out using IDBA-UD to assemble contigs. MetaBAT 2 [START_REF] Kang | MetaBAT, an efficient tool for accurately reconstructing single genomes from complex microbial communities[END_REF] was implemented for genome binning, with default settings. CheckM [START_REF] Parks | CheckM: assessing the quality of microbial genomes recovered from isolates, single cells, and metagenomes[END_REF] was then used to check the quality of metagenome assembled genomes (MAGs). Low quality MAGs, i.e. < 80% completeness and/or > 10% contamination, were removed from downstream analysis. FastANI [START_REF] Jain | High throughput ANI analysis of 90K prokaryotic genomes reveals clear species boundaries[END_REF] was used to assign taxonomy to the MAGs.

Sugars, ethanol and acetic acid quantification

Sugar consumption and metabolites production were analyzed in the liquid phase by ultra-performance liquid chromatography (UPLC-RI) (Thermo Scientific, Dardilly, France) according to the method described by [START_REF] Villarreal-Soto | Impact of fermentation conditions on the production of bioactive compounds with anticancer, anti-in fl ammatory and antioxidant properties in kombucha tea extracts[END_REF]. Analysis ranges varying from 1.25-10.00 g/L were used for the quantification of sucrose, glucose, fructose and ethanol as standards solutions and for glycerol and acetic acid a range from 0.25-2.00 g/L was used.

Liquid-liquid extractions and biological assays

Sequential liquid-liquid extractions with organic solvents (ethyl acetate and butanol) were performed with the kombucha samples at the end of the fermentation. The solvents were then evaporated using a vacuum rotary evaporator at 35 °C (RV 10 Auto VWR, IKA, Staufen, Germany) in order to obtain a dry extract.

Stock solutions were prepared from each dry extract in a concentration of 3 mg/mL, using DMSO 100% as solvent. Then, for all biological evaluations the stock solutions were tested initially at 50 μg/ mL and at 25 μg/mL or 5 μg/mL in the case of high inhibition percentages (> 70%) in order to calculate the IC 50 using the linear Watawana et al., 2015aWatawana et al., , 2015b) sources of carbon used [START_REF] Reiss | Influence of different sugars on the metabolism of the tea fungus[END_REF][START_REF] Vohra | Effects of medium variation and fermentation time on the antioxidant and antimicrobial properties of Kombucha[END_REF]Watawana et al., 2015aWatawana et al., , 2015b) ) and fermentation parameters [START_REF] Cvetković | Specific interfacial area as a key variable in scaling-up kombucha fermentation[END_REF][START_REF] Villarreal-Soto | Impact of fermentation conditions on the production of bioactive compounds with anticancer, anti-in fl ammatory and antioxidant properties in kombucha tea extracts[END_REF]. As a result, the fermented teas can have quite different properties and chemical compositions. While all of these aforementioned parameters considerably influence the bioprocessing of kombucha tea, these factors have in general been studied in isolation, and thus the relationship between bioactive molecule production and microbial metabolism has, to date, not been fully understood. Developing a deeper understanding of the links between all of these elements can be of considerable value with respect to optimizing the fermentation process and its final functionality. In this study, three different kombucha consortia were used to investigate their kinetics, microbial diversity via high-throughput sequencing, chemical composition and respective functional profile. This research is of relevance to kombucha fermentation globally, generating knowledge that will facilitate optimization of the process to yield desirable end products.

Materials and methods

Starter cultures

The kombucha pellicles used in this study were obtained from different origins. The first was obtained from local people of Toulouse, France (F), the second, from a commercial bottle of kombucha tea and reconstituted as described in Section 2.2 (G), and the third one was bought from the website www.jemangevivant.com (H). The choice of the suppliers was done randomly in order to study the impact of the specific microbiota over the biological profile of the fermented teas and because of its availability. All the pellicles were fermented three times before experimental use in order to stabilize the microbial consortium. The protocol described below is used in order to preserve the pellicles as a stock for later experiments.

Preparation of kombucha tea

Black tea was prepared according to the following procedure: 70 g of sugar were added to 1 L of water at 40 °C. Once the water reached 80 °C, 10 g of black tea (Royal Ceylan, Lipton ®) was added and allowed to infuse for 15 min. The tea was then removed and the infusion was left to cool. After the infusion was cooled to room temperature (25 °C), tea was poured into 2 L glass containers (ratios: s/h = 9.02 cm, s/ v = 0.132 cm -1 ) and 20 mL/L of the previous fermented liquid media, 10 mL/L of cider vinegar and 20 g/L of pellicle were added. The vessels were covered with cheesecloth and incubated at 25 °C for 15 days. All fermentations were performed in duplicate.

Shotgun metagenomic analysis

Samples for DNA extraction were taken after 15 days from both the tea and the pellicle. The Geneall Exgene Soil SV extraction kit was used for all extractions, with some modifications. For the pellicle DNA extraction, 0.8 g were removed and washed twice in 500 μL of sterile water, cut into smaller pieces and placed in a powerbead tube. Then, 550 μLof SL buffer, 90 μL of lysozyme (50 mg/mL) and 50 μL of mutanolysin (100 μL/mL) were added. The mixture was incubated at 60 °C for 15 min with occasional vortexing. Twenty-eight μL of proteinase K were then added, with further 15 minute incubation, including occasional vortexing. The tube was then placed in a bead beater (Qiagen Tissue lyser 2) for 10 min at 20 oscillations/s. The manufacturer's instructions were then followed, including pelleting and washing steps, with a final volume of 30 μL of elution buffer added at the final step.

Sample DNA extractions were performed by resuspending kombucha tea sample by stirring and 50 mL of tea were collected in a 50 mL falcon tube. Then the tube was centrifuged at 5000 rpm for 10 min at room temperature. The supernatant was discarded and the pellet was = -%inhibition 100 (Abs(control) Abs(sample))/Abs(control)

Anti-inflammatory activity

This biological assay was performed against the enzyme 15-LOX using a previously described method [START_REF] Villarreal-Soto | Impact of fermentation conditions on the production of bioactive compounds with anticancer, anti-in fl ammatory and antioxidant properties in kombucha tea extracts[END_REF]. One hundred and fifty microliters of 100 mM phosphate buffer (pH 7.4), 20 μL of the extract solution at a concentration of 3 mg/mL, 60 μL of linoleic acid (3.5 mmol/L), and 20 μL of 5-LOX (Soybean 500 U) were placed in a 96-well plate. The mixture was then incubated at 25 °C for 10 min and the absorbance was measured at 234 nm. The anti-inflammatory activity was defined as the percentage of inhibition of the 15-LOX enzyme. Nordihydroguaiaretic acid (NDGA) was used as a standard.

Antiproliferation evaluation

Antiproliferation test of kombucha extracts on human colon cancer (HCT-116) and human breast cancer (MCF7) cell lines was estimated as described previously [START_REF] Villarreal-Soto | Impact of fermentation conditions on the production of bioactive compounds with anticancer, anti-in fl ammatory and antioxidant properties in kombucha tea extracts[END_REF]. Cancer cells were maintained at 37 °C in a humidified 5% CO2 incubator (NBS Eppendorf, Germany) using Dulbecco's modified Eagle's medium (DMEM, Sigma Aldrich, USA) for the human breast cancer cell line and RPMI-1640 (Sigma Aldrich, USA) for the colon cancer cell line, respectively. Cells were then seeded in 96-well plates at a concentration of 3 × 10 4 cells/ well in 100 μL of culture medium, and then 100 μL of culture medium containing each sample at different concentrations were added. Nontreated cells were used as a control for the growth inhibition. The plates were incubated at 37 °C for 48 h, then the supernatant was then removed and 50 μL of MTT solution were added followed by an incubation for 40 min. After removing the MTT reagent (Sigma, M-5655), 80 μL of DMSO were added to solubilize the formazan crystals. The absorbance was measured at 605 nm. All extracts were re-suspended in DMSO followed by dilution in the buffer so that the DMSO did not exceed 1% as cells continue to multiply at this concentration. Tamoxifen was used as a positive control.

Phenolic and aromatic compounds determination

The dried extracts were injected at a concentration of 20 mg/mL of acetonitrile and water (20:80 v/v) and then phenolic and aromatic compounds were identified and quantified by HPLC-DAD (Thermo Scientific Dionex Ultimate 3000). Standard solutions were separately prepared at concentrations of 50, 20, 10, 5, and 1 mg/L using a (80:20 v/v) water/acetonitrile solution. Chemical composition was performed by gas chromatography-mass spectrometry (GC-MS) Varian Saturn 2000 (Les Ulis, France). Total phenolic composition was quantified using the Folin-Ciocalteu assay.

Results and discussion

Fermentation kinetics of different consortia and their produced metabolites

Sugar consumption and the production of the primary metabolites The non-fermented tea (NF) was also evaluated.

Antioxidant activity

The DPPH assay was measured according to a previously described method [START_REF] Villarreal-Soto | Impact of fermentation conditions on the production of bioactive compounds with anticancer, anti-in fl ammatory and antioxidant properties in kombucha tea extracts[END_REF]. Twenty microliters of the Kombucha fermented extracts were mixed with 180 μL of a 0.2 mM methanolic DPPH solution. The reaction mixture was then incubated at 25 °C for 30 min. A control experiment was also performed using ascorbic acid as a standard with the same procedure without the sample and the absorbance was read at 520 nm. The free radical-scavenging activity of each sample was then calculated by using the following formula: across the respective kombucha fermentations were analyzed. Despite the fermentation conditions remaining constant, differences in the specific microbial populations present had an impact on the kinetics and evolution of the process. Differences in the levels of sugar conversion were observed between days 0 and 3 (Fig. 1), where a 10 fold difference was apparent between the samples. The concentration of residual sugar was greatest for consortium H (~35 g/L), which may explain why ethanol production was lowest (6 g/L) in this sample after 15 days of fermentation. Sucrose was hydrolyzed into glucose and fructose in all cases. However, the manner in which they were consumed differed (Fig. 2). In sample F, sugars were completely depleted at 12 days, while for kombuchas G and H, sugars remained in the media. As noted kombucha H was extreme in this regard. More specifically, in this case fructose reached 12 g/L after 15 days. A similar sugar profile was observed by [START_REF] Chen | Changes in major components of tea fungus metabolites during prolonged fermentation[END_REF] and [START_REF] Kallel | Insights into the fermentation biochemistry of kombucha teas and potential impacts of kombucha drinking on starch digestion[END_REF] were fructose was poorly metabolized compared to glucose, probably because the latter is used for the biosynthesis of cellulose and for the production of organic compounds, such as gluconic acid, or because it is preferred as a carbon source by the specific kombucha microbiota present. However, it should be noted that in another kombucha study, [START_REF] Sievers | Microbiology and fermentation balance in a kombucha beverage obtained from a tea fungus fermentation[END_REF] observed that fructose was metabolized prior to glucose, again possibly due to differences in the specific microbial composition and/or initial carbon source [START_REF] Malbaša | Effect of sucrose concentration on[END_REF]. Acetic acid concentrations were similar across the three samples regardless of sugar consumption or ethanol production, reflecting the ability of the microbial populations present to use several metabolic pathways to produce acetic acid [START_REF] Gomes | Acetic Acid Bacteria in the Food Industry : Systematics[END_REF].

Metagenomics of solid and liquid phases

Shotgun metagenomics was used to examine the microbiota of these three kombucha communities (solid and liquid phases) after 15 days (Fig. 3). The relative abundance of bacteria was 2-fold higher in the pellicle than in the liquid phase with the relative abundance of yeast being highest in the liquid phase in all cases. Actinobacteria and Proteobacteria were identified in all samples, with Acetobacteraceae being the dominant family. In the case of the yeasts, the dominant taxa belonged to the Saccharomycetaceae and Schizosaccharomycetaceae families, both of which correspond to the phylum Ascomycota.

Microbial taxonomy

Almost 80% of the microbes present were Acetobacteraceae, with the dominant genera being Komagataeibacter, Gluconacetobacter and Gluconobacter (Fig. 4). Several species from the genus Acetobacter genus were also identified (A. malorum, A. pasteurianus, A. pomorum, and A. tropicalis). More specifically, at the species level, Komagataeibacter rhaeticus was the dominant bacteria (16-49%), followed by Gluconacetobacter sp. SXCC1 (9-26%), both being present in greatest proportions in the solid phases (Fig. 3). Komagataeibacter xylinus had a higher abundance in the liquid (16.89%) of kombucha G. Notably, it has been noted that strains from this species have an ability to survive gastric conditions, such as low oxygen pressure and the presence of bile salts and acidity [START_REF] Lavasani | Komagataeibacter xylinus as a novel probiotic candidate with high glucose conversion rate properties[END_REF]. Fig. 4 also shows the quantity of sugars, acetic acid, glycerol and alcohol for the three consortia. The most notable difference between the three liquid phase compositions is the low levels of Schizosaccharomycetaceae (S. pombe at species level) in H, which corresponds to the highest levels of all sugars left at the end of the experiment.

Four genera of yeast, i.e., Candida arabinofermentans, Brettanomyces bruxellensis, Schizosaccharomyces pombe and Zygosaccharomyces bailii, were identified across the three kombuchas.

The dominant fungal species, found primarily in the liquid phases, were B. bruxellensis (29.56%) and S. pombe (16.11%). Similar results were obtained by [START_REF] Coton | Unraveling microbial ecology of industrial-scale kombucha fermentations by metabarcoding and culture-based methods[END_REF], who revealed that their relative abundance changed with time. At day zero, most of the yeasts were found in the solid phase while at day 8 their abundance in the liquid increased. The same pattern was observed in this study, where yeasts proportion was higher in the liquid phases after 15 days of fermentation. From the perspective of individual kombuchas, sample F had the highest proportion of S. pombe and B. bruxellensis in the liquid phase. This may explain it having the fastest rate of sugar consumption (Fig. 2) and highest levels of ethanol production (17 g/L). However, B. bruxellensis was not detected in the liquid or solid phase of sample G, which had the slowest rate of sugar consumption and hydrolysis. As noted, even though Gluconacetobacter and Brettanomyces typically dominate the kombucha microbiome, several studies have highlighted variability [START_REF] Coton | Unraveling microbial ecology of industrial-scale kombucha fermentations by metabarcoding and culture-based methods[END_REF][START_REF] Marsh | Sequence-based analysis of the bacterial and fungal compositions of multiple kombucha (tea fungus) samples[END_REF][START_REF] Reva | Metabarcoding of the kombucha microbial community grown in different microenvironments[END_REF][START_REF] Teoh | Yeast ecology of kombucha fermentation[END_REF].

Salmonella enterica-associated reads were assigned by Kaiju in all 3 liquid samples and one solid sample, H, at abundances from 2.6% to 4.6%. While Kaiju was chosen as it outperformed other classifiers (Kraken2 [START_REF] Wood | Kraken: ultrafast metagenomic sequence classification using exact alignments[END_REF] and Bracken [START_REF] Lu | Bracken: estimating species abundance in metagenomics data[END_REF]) with respect to assigning the eukaryotic-associated reads, a vital consideration with respect to kombucha fermentation, Kaiju's reliance on the NR database, which has a disproportionate amount of pathogenic taxa due in part to their importance to science and medicine. Notably Salmonella was undetected above the 0.1% threshold by other classifiers and no Salmonella metagenome-associated genomes (MAGs) were generated. Metagenome-Assembled Genomes (MAGs) were also assembled for all kombucha samples. Seventeen medium quality MAGs were produced in total (above 80% complete, less than 10% contamination). These MAGs were all members of the Gluconobacter, Komagataeibacter and Pseudomonas genera with no MAGs being assembled for Salmonella. This adds further evidence that Salmonella was not in the samples, but Fig. 2. Evolution of average hydrolysis (left) and sugars consumption (right) rates for the three fermentations of kombucha. some of the short reads (prior to assembly) were being mapped to Salmonella due to the large number of Salmonella genomes in the NR database.

Functional profile

The functional profile of the three kombucha samples, as assessed by SuperFocus, was relatively homogenous. However, some differences were observed (Fig. 5). The kombucha H microbiota had the lowest proportion of genes associated with carbohydrate metabolism and respiration, reflecting the associated taxonomy and possibly explaining the higher quantities of sugar remaining in this sample. A deeper analysis (supplementary) reveals that, more specifically, kombucha H had less pathways involved in fermentation and central carbohydrate metabolism than kombucha F and G. Kombucha G had the highest abundance of pathways involved in sugar alcohols and was also the sample in which ethanol concentrations were highest at the end of the fermentation. Other interesting differences from the perspective of kombucha manufacturing include several amino acid synthesis pathways (such as the superpathway responsible for branched amino acid biosynthesis, ~0.2% higher in Kombucha H than both other samples) and the highest abundance of acetyl-CoA biosynthesis in Kombucha H (~0.5% higher than both F & G). Analysis of the data using Melonnpan was also performed. Melonnpan is a software tool used to predict the potential metabolites of a microbiome. Although many metabolites were measured (see subsequent sections), Melonnpan had the potential to predict if other desirable metabolites are produced in the samples. The predicted metabolites from this analysis were similar across the pellicle and tea samples from all kombuchas (Fig. 6). A secondary bile acid, deoxycholic acid, was predicted to be the most abundant metabolite in all cases. Deoxycholic acid is important in both human health research [START_REF] Deeks | Deoxycholic acid: a review in submental fat contouring[END_REF] and in industry [START_REF] Kim | Physicochemical characterizations of self-assembled nanoparticles of glycol chitosan-deoxycholic acid conjugates[END_REF]. Its use as an emulsifier of fats in particular is driving the growth of its value globally. Glutamate was predicted to be the next most abundant metabolite. Glutamate is a neurotransmitter and is known for its role in mental health [START_REF] Cryan | A glutamate pathway to faster-acting antidepressants? Science[END_REF]. The profile also includes many short chain fatty acids, polyunsaturated fatty acids, other bile salts, vitamins (mostly B vitamins), and volatile compounds.

HPLC analysis of phenolic compounds

Polyphenols are major plant secondary metabolites that exhibit a remarkably diverse range of bio-physicochemical properties, and are ubiquitous in plant extracts [START_REF] Quideau | Plant polyphenols: chemical properties, biological activities, and synthesis[END_REF]. HPLC analysis was performed in order to identify the main phenolic compounds produced in the 3 kombucha beverages (Table 1). Gallic acid was produced by the three consortia in similar concentrations. This is consistent with the presence of genes encoding the enzyme responsible for gallic acid production, i.e., shikimate dehydrogenase, across all three microbiomes. Catechin and epicatechin decreased after fermentation in all cases. [START_REF] Jayabalan | Changes in content of organic acids and tea polyphenols during kombucha tea fermentation[END_REF] observed the same pattern when studying the changes in tea polyphenols during kombucha fermentation and attributed the phenomenon to biodegradation by enzymes secreted by yeasts and bacteria. This decrease was also observed by [START_REF] Payne | Impact of fermentation, drying, roasting, and Dutch processing on epicatechin and catechin content of cacao beans and cocoa ingredients[END_REF] when studying the fermentation of cacao beans but, in contrast, a previous kombucha study [START_REF] Villarreal-Soto | Impact of fermentation conditions on the production of bioactive compounds with anticancer, anti-in fl ammatory and antioxidant properties in kombucha tea extracts[END_REF] noted an increase in catechin concentrations after a 21 day fermentation when a higher surface/height ratio was employed. Caffeic acid, previously observed to be produced from the microbial conversion of other phenolic acids [START_REF] Lin | Biosynthesis of caffeic acid in Escherichia coli using its endogenous hydroxylase complex[END_REF], increased after fermentation. Some observations related to specific kombuchas only, for example, theobromine, rutin and chlorogenic acid production was favored by consortium G. It was also noted that only trace amounts of coumaric, ferulic and trans-cinnamic acids were found after fermentation across all three kombuchas. This may be due to the ability of Brettanomyces bruxellensis to catalyse the transformation of ρ-coumaric and ferulic acids into vinyl-and ethyl-phenols [START_REF] Romano | Growth and volatile compound production by Brettanomyces/Dekkera bruxellensis in red wine[END_REF]. Fig. 7 shows the high-resolution separation of 22 compounds in the ethyl acetate extracts of the three different consortia. A number of these compounds were identified and quantified as 3-gallic acid; 13-chlorogenic acid; 18coumaric acid; 19-ferulic acid; 20-taxifolin and 22-trans-cinnamic acid (Table 1). When comparing patterns to those of non-fermented tea, it was observed that several compounds were transformed. Moreover, compounds 2, 8 and 12 were produced after fermentation, but their presence varied depending on the kombucha tested.

As depicted (Fig. 7), a number of notable differences were observed in the case of kombucha F, with gallic acid and compounds 4 and 5 greatly increasing after fermentation and compound 9 being unique to this community. In addition, peak "a" was completely transformed after fermentation of kombucha F, but remained in the other two kombuchas.

Gas chromatography-mass spectrometry analysis of kombucha metabolites

GC-MS also contributed to metabolite analysis and led to the identification several organic acids, sugars, alcohols and phenolic compounds (Table 2). Before derivatization, 2,2-methylenebis(4-methyl-6-tert-butylphenol) was the only compound detected in the ethyl acetate extracts from any of the three kombuchas or the non-fermented tea. In order to improve the identification and volatility of the produced compounds, silylation was performed, leading to the detection of twenty-six compounds. Organic acids such as isovaleric, succininc, malic, pentanoic and citric were predominantly found, in similar proportions, in kombuchas F and H, but propanoic acid was present in 4-fold higher abundance in sample (H). Kombucha G had the lowest levels of organic acids, corresponding to the absence of B. bruxellensis [START_REF] Andrade | Influence of Dekkera bruxellensis on the contents of anthocyanins, organic acids and volatile phenols of Dao red wine[END_REF]. Succinic acid is a dicarboxylic acid produced mainly as an intermediate of the tricarboxylic acid cycle during aerobic respiration and is also one of the fermentation end products of anaerobic metabolism [START_REF] Chidi | Organic acid metabolism and the impact of fermentation practices on wine organic acid metabolism and the impact of fermentation practices on wine acidity : a review[END_REF]. The formation of organic acids, such as succinic acid, is reported to vary significantly with different fermentation conditions [START_REF] Lamikanra | Changes in organic acid composition during fermentation and aging of noble muscadine wine[END_REF]. Its production also varies according to the microbial population, as was also observed in this study. L-(-)-Sorbofuranose was only detected in Kombucha F, but several sugars were unique to kombucha H, which was also the kombucha with the highest concentration of residual sugars after fifteen days of fermentation. 2,3 butanediol, 2-phenylethanol, glycerol and 2-(4-hydroxyphenyl) ethanol were detected in all the samples, with the exception that 2-phenylethanol was not detected in kombucha G. The greatest proportions of these compounds were produced in kombucha F, which was also the fermented tea with the highest level of ethanol production (18 g/L). Among other detected compounds, all of the produced phenolic compounds were present in the three kombuchas in similar proportions, except from catechin, which was only detected in kombuchas F and H. Results from a previous study [START_REF] Villarreal-Soto | Impact of fermentation conditions on the production of bioactive compounds with anticancer, anti-in fl ammatory and antioxidant properties in kombucha tea extracts[END_REF] showed higher quantities of glycerol production compared to those observed here, possibly as a consequence of the differences in the fermentation kinetics. Even if most of the yeast species have similar central carbon metabolic pathways, differences in nutrient uptake and utilization, as well as in the regulation of fermentation and respiration, may yield different secondary metabolites [START_REF] Chidi | Organic acid metabolism and the impact of fermentation practices on wine organic acid metabolism and the impact of fermentation practices on wine acidity : a review[END_REF] Fig. 6. Melonnpan results showing the top 50 most abundant predicted metabolites from F, G and H. Both the predicted metabolites from the SCOBY and Tea sequences are shown in the plot.

Biological evaluation of Kombucha teas

In the interest of evaluating the beneficial potential of the fermented teas different in-vitro assays were performed.

Antioxidant capacity and total phenolic composition

As observed previously, similar concentrations of phenolic compounds were obtained in the non-polar extracts of all our samples (Fig. 8). Ethyl acetate has also been reported to extract the highest amount of total phenolic compounds and total flavonoids in some previous studies [START_REF] Boussoussa | Effect of different solvent polarity on extraction of phenolic compounds from Algerian Rhanterium adpressum flowers and their antimicrobial and antioxidant activities[END_REF][START_REF] Rahmani | Kombucha fermentation of African mustard (Brassica tournefortii) leaves : chemical composition and bioactivity[END_REF]. Gallic acid showed its highest concentration in the ethyl acetate extracts according to the HPLC-DAD analysis. This acid has been found to exhibit the greatest antioxidant capacity among various polyphenols [START_REF] Badhani | Gallic acid : a versatile antioxidant with promising therapeutic and industrial applications[END_REF], which may explain why the highest inhibition percentage against the DPPH radical was achieved with the non-polar extracts obtained in this study. The attained inhibition percentages are higher than those reported by other authors [START_REF] Malbaša | Influence of starter cultures on the antioxidant activity of kombucha beverage[END_REF][START_REF] Vohra | Effects of medium variation and fermentation time on the antioxidant and antimicrobial properties of Kombucha[END_REF], who established around 60% of inhibition after 7-10 days of fermentation. The antioxidant activity of the phenolic acids is affected by several factors such as the phenolic aromatic arrangement or the position of the hydroxyl group [START_REF] Badhani | Gallic acid : a versatile antioxidant with promising therapeutic and industrial applications[END_REF]. In addition, the extraction of the phenolic compounds depends on their polarity and the affinity for the organic solvent. This could explain the difference in the total phenolic compound composition and consequently in the antioxidant activity of the butanolic extracts and aqueous phases. It can be observed (Fig. 8) that the phenolic composition of the ethyl acetate extracts is 2-fold higher than that of the butanolic extracts. Nevertheless, the extraction yields of the ethyl acetate extracts are lower than those of the butanolic extracts (Fig. 9). Both extracts exhibit similar inhibition percentages against the free radicals at 50 μg/mL. A higher concentration of its IC 50 value, of 16 μg/mL was obtained with the butanolic extracts compared to 7 μg/mL with the ethyl acetate extracts (Table 3). Suggesting that the non-polar phenolic compounds are more stable and thus have a higher antioxidant activity. The higher antioxidant activity of the ethyl acetate extracts could be related to the higher presence of gallic acid in the three consortia (11,997.7-13,020.9 mg/kg of dry extract), however no other compound was detected in similar concentrations in the butanolic extracts, which also showed a good antioxidant activity. This suggests that there may be other phenolic compounds present, responsible for preventing the oxidation that were not quantified in this study.

Anti-inflammatory activity

The anti-inflammatory activity of kombucha tea, as revealed by in vitro assays, has been reported previously [START_REF] Vázquez-Cabral | Oak kombucha protects against oxidative stress and inflammatory processes[END_REF][START_REF] Villarreal-Soto | Impact of fermentation conditions on the production of bioactive compounds with anticancer, anti-in fl ammatory and antioxidant properties in kombucha tea extracts[END_REF], highlighting improved inhibition of some enzymes responsables of inflammation by kombucha relative to unfermented tea. It is thought that the bioactivity of the final product is directly related to the processing technologies and its microbial and chemical composition [START_REF] Michalska | Bioactive compounds of blueberries : post-harvest factors influencing the nutritional value of products[END_REF]. In the present study, it was observed (Fig. 10) that the highest anti-inflammatory activity after fermentation was obtained with kombucha G with an inhibition percentage of 44.5%, followed by 28% with consortium (H) in the ethyl acetate extracts. These results confirmed that kombucha consortia have anti-inflammatory potential against the 15-lipoxygenase enzyme.

Antiproliferative activity on human tumor cell lines

The anti-cancer effect of kombucha extracts against the human cancer cell lines HCT-116, MCF-7 and OVCAR was investigated. The samples showed a higher selective anti-proliferative activity against HCT-116 line in the case of the butanolic extracts compared to the nonfermented tea (Table 4). In particular, kombucha G extracts seemed to best inhibit this cell line, with a 50% increase in inhibition being observed with the ethyl acetate and butanol extracts from the 21 day of

Table 1

Content of phenolic compounds, theobromine and caffeine from black tea (NF) and kombucha extracts by HPLC (mg/kg of dry extract). 2019), who found greater inhibition after fermentation reaching around 60%. In the case of the MCF-7 cancer cell line, kombucha F showed a slight increase in activity after 15 days of fermentation compared to the non-fermented tea. However, the remaining extracts did not show any increase in activity compared to the control. Moreover, kombucha extracts did not show cytotoxic activity against the OVCAR cancer cells. Indeed, in this instance cytoxicity decreased with fermentation.

Compound

Conclusions

The study of metagenomic DNA from the solid and liquid phases of three kombucha consortia revealed differences across kombuchas and between the two phases. Fermentation kinetics showed an association between the microbiota and sugar consumption as well as secondary metabolite production. Some similarities were observed between the three different microbial populations. The most abundant species of bacteria were the same in all the samples, differing only in relative abundance. The yeast populations differed most considerably and, as a consequence, the sugar profile differed, in a manner that reflected the presence or absence of S. pombe. Microbiota differences also corresponded with difference in the biological profiles of the obtained teas, with extracts from kombucha G, which contained the highest relative abundance of Komagataeibacter xylinus in both solid and liquid phases and Schizosaccharomyces pombe in the liquid phase, demonstrating the greatest anti-inflammatory and anti-proliferation activities. IC 50 values were calculated for inhibition percentages higher than 70% at 25 and 5 μg/mL ± standard deviations (n = 3).

Table 2 GC-MS analysis (area x10 6 ) of kombucha tea samples and black tea (NF) without and with derivatization.

Fig. 1 .

 1 Fig. 1. Evolution of the main compounds during kombucha fermentations. All the data are average values of triplicate analysis. Sucrose left Y-axis, Glucose, Fructose, Glycerol, Ethanol and Acetic acid, right Y-axis.

Fig. 3 .

 3 Fig. 3. Taxonomic classification of microbial population in the solid and liquid phases of the three different consortia after 15 days of fermentation.

Fig. 4 .

 4 Fig. 4. Most abundant species and families across F, G and H. Sugars, glycerol, acetic acid and ethanol concentrations at the endpoint of the three consortia.

Fig. 5 .

 5 Fig. 5. Differences in the SuperFocus Level 1 pathways. A) Shows what pathways G and H have a higher relative abundance of (in red), or lower relative abundance of (in blue) than sample F. B) Shows samples F and H compared to G, and C) shows samples F and G compared to H. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

  the non-fermented tea. This antiproliferative effect against human colon cancer cells has been previously reported byJayabalan et al. (2011) and Villarreal-soto et al. (

Fig. 7 .

 7 Fig. 7. HPLC chromatograms of (a) ethyl acetate and (b) butanol kombucha extracts from the three consortia.

Fig. 8 .Fig. 9 .

 89 Fig. 8. Extraction of different solvents and residual aqueous phases from the three different consortia.

Fig.

  Fig. Anti-inflammatory activity of black tea (NF) and three kombucha samples. All the data are average values of triplicate analysis.

Table 3

 3 Half-maximal inhibitory concentration of the antioxidant activity of three kombucha consortia and black tea (NF).

	Solvents	Samples IC 50 (μg/mL)
		NF	F	G	H
	EtOAc	7 ± 0.2	7.0 ± 0.4	6 ± 0.3	8 ± 0.9
	BuOH	30 ± 0.5	12 ± 0.3	13.0.1	24 ± 0.8
	H 2 O	> 50	> 50	> 50	> 50

Table 4

 4 Antiproliferation activity against human colon cancer (HCT-116), human breast cancer (MCF-7) and human ovarian cancer (OVCAR) cell lines.All the data are average values of triplicate analyses. Where NF: black tea, N.A: not active.

	Sample	Extract	Inhibition % at 50 μg/mL
			HCT-116	MCF-7	OVCAR
	NF	EtOAc	8.0 ± 0.7	14.6 ± 0.2	34.7 ± 4.7
		BuOH	4.4 ± 2.0	N.A	16.5 ± 2.0
		H 2 O	N.A	14.0 ± 0.4	14.6 ± 1.2
	F	EtOAc	10.3 ± 0.4	6.9 ± 0.7	N.A
		BuOH	13.8 ± 0.7	4.5 ± 1.6	N.A
		H 2 O	1.7 ± 0.9	18.0 ± 1.4	N.A
	G	EtOAc	16.3 ± 1.8	7.8 ± 0.2	29.1 ± 2.3
		BuOH	10.5 ± 1.1	3.0 ± 0.3	1.1 ± 0.3
		H 2 O	N.A	15.3 ± 0.6	N.A
	H	EtOAc	13.7 ± 1.1	10.5 ± 0.9	N.A
		BuOH	8.2 ± 0.2	N.A	N.A
		H 2 O	N.A	N.A	N.A

Acknowledgments

The authors thank the Mexican Council of Science and Technology (CONACYT-México) for funding the doctoral scholarship. In addition, they thank the TEAGASC research center for the facilities, Paul Cotter for reviewing the manuscript and John Leech for the bioinformatics analysis and interpretations. The metagenomics and bioinformatics analysis were conducted with the financial support of Science Foundation Ireland (SFI).

Ultimately, this study employed a variety of approaches to investigate the fermentation profiles, microbiota, metabolites and health promoting potential of a variety of kombucha teas. These insights will prove valuable with respect to the optimization of the microbiota of kombuchas with respect to industrial production and health promoting attributes.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.


Coralie Laveau
email: coralie.laveau@agro-bordeaux.fr

Séverine Mary

Guillaume Darrieutort

Cornelis Van Leeuwen

Maarten Van Helden

Impact of sown fallows on the Xiphinema index populations in different soil types

Keywords: Fallow, Xiphinema, Virus, Grapevine fanleaf, soil type

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Fanleaf degeneration is a viral disease affecting the vines longevity, the production potential and the quality of wine grapes. Two-third of French Vineyards is affected by this viral disease [START_REF] Demangeat | Le court-noué de la vigne: l'état des connaissances sur cette maladie lors de la journée "Alternative" du 28 avril 2005[END_REF]. The two main viruses responsible for the fanleaf disease are the grapevine fanleaf virus (GFLV) and the arabic mosaic virus (ArMV). They are respectively transmitted by Xiphinema index and Xiphinema diversicaudatum which often living deep in the soil [START_REF] Andret-Link | Grapevine fanleaf virus : still a major threat to the grapevine industry[END_REF]. Among the virus vector's nematodes, X. index has the most significant economic impact. The worldwide distribution of X. index is closely related to that of grapevine. The propagation of GFLV is possible either by plant material or by nematodes from grapevine to grapevine. As the dissemination by propagation material has been reduced by clonal selection and certification for plants, it is now necessary to find strategies to control the propagation of GFLV in vineyard conditions. An effective strategy is needed to reduce nematode populations in the soil. For a long time, winegrowers used chemical products to fumigate the soil between two successive grapevine crops. One of the alternatives to the use of these toxic synthetic products is the use of antagonistic plants during fallow period for their effect on the reduction of nematodes vectors. Several plants are known to their nematicidal effect on different crops. For instance, Tagetes spp. have been identified to their effective control of some species of root-infesting nematodes as Pratylenchus penetrans or Meloidogyne hapla [START_REF] Oostenbrink | Tagetes patula L. als voorvrucht van enkele land-en tuinbouwgewassen op zand-en dalgrond[END_REF][START_REF] Hijink | Influence of different compositae on population density of Pratylenchus penetrans and some other rootinfesting nematodes[END_REF]. Sorghum vulgare incorporated as green manure before planting lettuce resulted in lower reproduction of Meloidogyne hapla [START_REF] Viaene | Management of Meloidogyne hapla on Lettuce in organic soil with sudangrass as a cover crop[END_REF]. In vitro biotests, Thymus vulgaris or Ruta graveolens extracts showed a nematicidal activity against X. index and X. americanum [START_REF] Insunza | Nematicidal activity of aqueus plants extracts on Xiphinema index[END_REF] but this effect was not confirmed in field tests. Chenopodium ambrosioides extracts have an effect on X. index and X. americanum in vitro but also against Meloidogyne spp. or Ditylenchus dipsaci [START_REF] Espinoza | Chenopodium ambrosioides, planta con uso potencial en el combate de nematodos fitoparasitos[END_REF][START_REF] Insunza | Control of Ditylenchus dipsaci on garlic (Allium sativum) with extracts of medicinal plants from Chile[END_REF]. Aballay showed that all the plants tested in greenhouse trial (Chenopodium ambrosioides, Ruta graveolens, Thymus vulgaris and Brassica juncea) were able to significatively reduce X. index populations but the final densities were significantly higher than chemical control [START_REF] Aballay | Evaluation of five nematode-antagonistic plants used as green manure to control Xiphinema index Thorne et Allen on Vitis vinifera[END_REF]. Field tests with Brassicus napus, Cosmos bippinatus, Lupinus albus, Thymus vulgaris, Zinnia elegans, Hordeum vulgare or Calendula officinalis, showed none of these plants were able to control the population of nematodes during their development or incorporation. Only Brassica significantly decreased populations after incorporation [START_REF] Aballay | Evaluation of plants with nematicidal properties in the control of Xiphinema index on table grapes cv. Thompson Seedless in the central zone of Chile[END_REF]. This study evaluates the use of a few plant species, previously selected through greenhouse screening, for their capacity to reduce X. index populations [START_REF] Laveau | Management of grapevine fanleaf virus vectors[END_REF][START_REF] Villate | variabilité et gestion des populations de Xiphinema index, le nématode vecteur du Grapevine fanleaf virus (GFLV)[END_REF] during fallow in the field.

Material and method

Seven plant species able to decrease the nematodes populations by about 50% compared to bare soil (negative control) in controlled conditions [START_REF] Laveau | Management of grapevine fanleaf virus vectors[END_REF][START_REF] Villate | variabilité et gestion des populations de Xiphinema index, le nématode vecteur du Grapevine fanleaf virus (GFLV)[END_REF], were tested in field conditions. Several trials were carried out between 2007 and 2012 with the same plants

 Experimental field design

The trials were carried out in the Bordeaux and Burgundy vineyard regions. For each trial, the protocol design encompassed 10 to 20 replicates of each tested plant. In the chessboard-like arrangement, each replicate (3 x 3m plot) was surrounded by several control plots (negative control = bare soil). A total of 98 to 142 plots was required to evaluate 3 to 4 plants per trial. Mechanical weeding was performed on the bare soil plots. The sowing of the plant plots was carried out at the end of April for the first trial and in September/October for the four others trials with a SEMBONER manual single row seeder. The plants were grown from 4 to 9 month depending on sowing date and vine planting date. Table 1 shows the characteristics of each trial.  Nematodes sampling and extraction At each sampling point, a trench (1.5 m long, 0.4 m wide, 1 m deep) was dug with an excavating machine. A 4 liter sample of soil was collected manually from 5-6 points from the undisturbed sides of the trench at depths of 0.3 to 0.8 depending on soil profile. After mixing, nematodes were extracted in the laboratory from a 2 liter subsample, using the method described by Villate [START_REF] Villate | variabilité et gestion des populations de Xiphinema index, le nématode vecteur du Grapevine fanleaf virus (GFLV)[END_REF]. Adults and juveniles were counted from the final suspension under a dissecting microscope. Maximum observed rooting depth was observed for each plot  Data analysis For each site, the effect of sown fallows on the rooting depth was analyzed using ANOVA. When a significant effect was found, multiple comparisons were conducted to test differences using Tukey's HSD test.

For each site, number of X. index on soil and number of X. index on bare soil were defined as a matched pair of counts. Effect of plants on X. index were analyzed as proportion data using a GLM with binomial errors and logit link and to test the difference to zero. Soil effect was tested with mixed-model. We used fixed effects for plants and soils and a random effect to take in account the site.

Results

 Vine root system

To control X. index, the first challenge for the plants tested is to reach the deeper soil layers where the nematodes live. So rooting depth was measured for each plant on each plot. The results are presented in figure 1. In trial A, Medicago hybride and Tagetes minuta roots reached 75 cm mean depth whereas those of Lupinus albus and Vicia villosa reached only 38 cm. In trial B, there was no difference between rooting depths, Medicago hybride obtained 65 cm mean depth which was less than in trial A. For the same growing time, roots of Medicago hybride were even less developed in trial C and D with respectively 39 cm and 53 cm mean depth compared to the two first trials but Medicago was still the deepest rooting plant. In trial C, the difference was significant with Vicia villosa (32 cm) and Avena sativa (29 cm). In trial D, Onobrychis viciifolia obtained results similar to Medicago hybride with 50 cm mean depth while roots of Avena sativa reached 43 cm . In the last trial E, the plants were poorly developed the day of the observations in April, their roots had still reached 20 cm mean depth for Vicia villosa and around 15 cm mean depth for Avena sativa and Medicago hybride. Based on these results, it seems that the rooting depth is more dependent on the soil type than the length of the crop cycle (except for test E). For the same species, rooting will be deeper in sandy gravel soils than in clay limestone soils.

 Antagonistic effect of plant species on

Xiphinema index numbers All tested plant were able to reduce the populations of X. index compared to bare soil during at least one trial (one vegetative cycle in field) except Trifolium pratense (Figure 2). However, there are differences depending on the test sites. In the first trial (Site A), sown in spring and harvested in August, revealed a good efficiency of all 4 plants. The strongest reduction of X. index populations was obtained with Tagetes minuta that reached more than 60% decrease in X. index. A decrease in X. index populations of 40% and 30% was observed on plots sown with Vicia villosa and Lupinus albus, respectively, whereas only 5% reduction on X. index populations was obtained for Medicago hybride compared to bare soil. But only Tagetes minuta and Vicia villosa show a significant difference compared to the bare soil. In the second trial (Site B) was sown in autumn and harvested the next spring, which prevented the sowing of (frost sensitive) summer crops Tagetes and Lupinus albus. Only Medicago hybride and Vicia villosa species could be evaluated again. Trifolium pratense and Avena sativa completed the design trial. The best result in this trial was obtained with Avena sativa which reached more than 40% decrease of X. index compared to bare soil but the difference was not significant. Vicia villosa and Medicago hybride were less successful with only 24 % decrease in X. index numbers for Medicago hybride and no significant difference with bare soil. Trifolium pratense, that gave very good results in the greenhouse, [START_REF] Laveau | Management of grapevine fanleaf virus vectors[END_REF][START_REF] Villate | variabilité et gestion des populations de Xiphinema index, le nématode vecteur du Grapevine fanleaf virus (GFLV)[END_REF] did not confirm this in the field. The other three trials were sown in October 2012 and harvested in late spring 2013. The trial on the site E was harvested earlier in April. On site C, the plants fallow did not reduce the populations of nematodes and on the contrary, there was more X. index than the bare soil control at the end of the trial (no statistical difference). On site D, all plants showed a decrease of X. index populations after 9 month in the same proportion with Avena sativa and Medicago hybride as in trial site B but the differences were significantes compared to the bare soil on this trial. Onobrychis viciifolia did not confirm in the field the results obtained in greenhouse. In site E the plants didn't have time to develop but Medicago hybride still allowed a very strong reduction in populations compared to the control with a statistical difference. The period of plant development does not seem to have a strong impact on the percentage reduction of X. index populations. On the other hand, soil type seems to play a larger role in the nematodes population decrease with a stronger decrease on sandy gravel soils than on clay limestone soils.

Discussion

All the plants tested in field conditions had shown reduced nematode populations in greenhouse tests but they did not confirm these results in all field trials. In the field, the highest reduction was obtained with Medicago sativa (trials B, D and E) but in trial A this plant showed no effect and in trial C there was more X. index on average on the plots of this plant compared to the bare soil plot. Conversely, Avena sativa gave the most regular results in the different trials with reduction between 30% and 45% on trials B and D but no effect of this plant compared to bare soil in trial C and E. Vicia villosa did not confirm the strong diminution of X. index populations compared to the bare soil, obtained in the first trial, in the next trials. The decrease of X. index populations were better in greenhouse than those in field for Tagetes minuta and a little less for Lupinus albus but these two plants have been set up on only one trial [START_REF] Laveau | Management of grapevine fanleaf virus vectors[END_REF]. However Tagetes spp. is known for its strong nematicidal effect on several species of nematodes and the results obtained in this test confirms once again this property. Lupinus albus reduced X. index population in our conditions while Aballay and Insunza [START_REF] Aballay | Evaluation of plants with nematicidal properties in the control of Xiphinema index on table grapes cv. Thompson Seedless in the central zone of Chile[END_REF] had not shown efficacy. Likewise, Trifolium pratense did not reduce populations of nematodes in the field, but was used in only one trial. We can not explain why Vicia villosa, Medicago hybride, and to a lesser extent Avena sativa, have led to an increased survival of the populations of nematodes on the site C in Burgundy. The implantation of more trials in this geographic area would have been necessary to conclude. It could be that anaerobic conditions due to waterlogging in the bare soil reduced nematode numbers, while waterlogging was prevented by the plants. The success of one of a plant should be connected with its development and particularly with its rooting depth. However, the best results have not always been obtained with the plants which had the deepest root system. Many unexplained points persist and the implantation of new trials is necessary to better qualify the link between the pedoclimatic conditions, the implantation of the species and their efficiency to reduce the populations of X. index. New experiments will be conducted to better understand the mechanisms of action of the best plants to reduce populations of X. index in the field. New trials will be set up to follow the rate of recontaminations of a new vine by the GFLV after two years of fallow with these nematicidal plants.

Figure 1 :

 1 Figure 1: Depth of rooting of each plant on the 5 test sites.

Figure 2 :

 2 Figure 2: Efficiency of fallow plants on the reduction of X. index populations compared to bare soil

Table 1 :

 1 Characteristics of the five trials

	Trial	A	B	C	D	E
	Site	Margaux (33)	Saint Emilion	Gevrey	Saint Emilion	Arsac (33)
			(33)	Chambertin (21)	(33)
	Climate	Oceanic	Oceanic	Continental	Oceanic	Oceanic
	Soil	Sandy gravel	Sandy gravel soil	Clay limestone	Clay limestone	Sandy gravel
		soil		soil	soil	soil
	Uprooting	March 2007	March 2009	May 2012	July 2012	March 2012
	date
		Vicia villosa,	Avena sativa,	Avena sativa,	Onobrychis	Avena sativa,
	Plants	Lupinus albus,	Vicia villosa,	Vicia villosa,	viciifolia, Avena	Vicia villosa,
		Tagete minuta,	Medicago	Medicago	sativa,	Medicago
		Medicago	hybride, Trifolium	hybride	Medicago	hybride
		hybride	pratense		hybride
	Control	Bare soil	Bare soil	Bare soil	Bare soil	Bare soil
	Number	10	12	20	20	20
	of
	replicates
	Samples	98	142	110	110	114
	number
	Sowing	April 2007	September 2009	October 2012	September 2012 October 2012
	Sampling	August 2007	July 2010	June 2013	June 2013	April 2013
	date
	Period	4	9	8	9	6

Acknowledgements

We thank the estates and their vineyard managers that allowed the realization of the trials on their plots and have funded the first one. We also thank France AgriMer that financially supported the three last trials.