PLANT MICROBE INTERACTIONS

New Insights into the Ecological InteractionBetween Grape Berry Microorganisms and Drosophila FliesDuring the Development of Sour Rot

André Barata & Sara Correia Santos &

Manuel Malfeito-Ferreira & Virgílio Loureiro

Received: 1 July 2011 /Accepted: 8 March 2012 /Published online: 22 March 2012# Springer Science+Business Media, LLC 2012

Abstract In this work, we studied the ecological interac-tions between grape berry microorganisms and Drosophilasp. flies involved in sour rot disease during grape ripening.After veráison the total microbial counts of grape berriesaffected by sour rot increased from about 2 log CFU/g ofberries to more than 7 log CFU/g. Berry damage provoked aclear shift in yeast diversity from basidiomycetes to asco-mycetous fermentative species. The latter were mostlyPichia terricola, Hanseniaspora uvarum, Candida zempli-nina, and Zygoascus hellenicus. However, these specieswere not able to produce the metabolites characteristic ofsour rot (gluconic and acetic acids) in inoculated berries. Onthe contrary, the acetic acid bacteria Gluconacetobactersaccharivorans produced high levels of these acids, mainlywhen berries were incubated in the presence of the insectDrosophila sp. Sour rot was not observed when grapebunches were physically separated from insects, even whenberries were artificially injured. The wounds made in berryskin healed in the absence of insects, thus preventing thedevelopment of sour rot. Therefore, in the vineyard, theinduction of sour rot depends on the contamination ofwounded berries by a microbial consortium—yeasts andacetic acid bacteria—transported by drosophilid insectswhich disseminate sour rot among damaged berries. In theabsence of these insects, plant defense mechanisms are

effective and lead to skin healing, preventing disease spread.Thus, we showed that Drosophila sp. act as a vector formicroorganisms associated with grape sour rot disease.

Introduction

Insects are probably the most significant source of yeaststhat can colonize grape berries [1]. There is a wide diversityof insects including honey bees, syrphid flies, wasps, moths,mites, mealybugs, weevils, cicadas, locusts, spiders, ear-wigs, and grasshoppers that visit, infect, and attack grapevines at various stages of their annual development [2]. It ismost likely that these “visitors” contribute to the yeast floraof grapes. In particular, drosopholid fruit flies, mainly Dro-sophila melanogaster (Diptera: Drosophilidae) commonlyvisit grapes to feed on the yeasts associated with grapeberries and identical species can be recovered from boththe fruit and the body of the insect [3].

The mutualistic association between Drosophila flies andyeasts occurs mainly on rotting fruits. Yeasts are the majornutritional source, providing proteins, vitamins, and othernutrients for Drosophila adults and their larvae while theflies disperse the yeasts among the different microhabitats[3–5]. The role of bacteria in Drosophila ecology has re-ceived less attention than that of yeasts. Recent research inmicrobe–insect symbiosis has shown that acetic acid bacte-ria (AAB) establish symbiotic relationships with severalinsects of the orders Diptera, Hymenoptera, Hemiptera,and Homoptera, all relying on sugar-based diets, such asnectars, fruit sugars, or phloem sap [6]. The fruit flies D.melanogaster hosts a rich AAB microbiome that have beenfound to be associated with the bacterial genera Acetobacter,Gluconacetobacter, Gluconobacter, and the novel genusCommensalibacter [7–11]. AAB establish symbiotic

Electronic supplementary material The online version of this article(doi:10.1007/s00248-012-0041-y) contains supplementary material,which is available to authorized users.

A. Barata (*) : S. C. Santos :M. Malfeito-Ferreira :V. LoureiroLaboratório de Microbiologia, Departamento de Recursos NaturaisAmbiente e Território, Centro de Botânica Aplicada à Agricultura,Instituto Superior de Agronomia, Technical University of Lisbon,Tapada da Ajuda,1349-017 Lisbon, Portugale-mail: abbarata@isa.utl.pt

Microb Ecol (2012) 64:416–430DOI 10.1007/s00248-012-0041-y

associations with the insect midgut, a niche characterized bythe availability of diet-derived carbohydrates and oxygenand by an acidic pH, selective factors that support theirgrowth. AAB have been shown to actively colonize differ-ent insect tissues and organs, such as the epithelia of maleand female reproductive organs and the salivary glands [6].

Fruit flies (Drosophila sp.) are constantly present in thevicinity of the grape bunches infected by sour rot, in everystate of development (eggs, larvae, pupae, and adults) andare empirically regarded as the most important vectors ofsour rot agents [12, 13]. This grape disease affects late-ripening cultivars with tightly packed, thin-skinned, anddense bunches close to harvesting, causing heavy crop los-ses and is detrimental to juice and wine quality [14–17].Sour rot is mainly induced by yeasts [18, 19] associated withAAB (Acetobacter spp. and Gluconobacter spp.) [12, 20,21]. Fruit flies captured in vineyards affected by sour rot arecarriers of yeasts (Kloeckera apiculata, Candida stellata,Pichia membranifiaciens, Metschnikowia pulcherrima, C.krusei) and AAB (Gluconobacter sp. and Acetobacter pas-teurianus), which are assumed to be implicated in sour rot[22].

Despite this knowledge, the role of yeasts and AAB onthe development of sour rot and the contribution of Dro-sophila flies on the grape decay process has not been inves-tigated to any great extent. Therefore, the purpose of thiswork was to find the main microbial agent of sour rot andthe importance of the insects in the development of thedisease.

Material and Methods

Vineyard Experimental Trials

During the 2008 vintage, the parcel of Vitis vinifera L. cv.Trincadeira red grape variety (1,500 m2; 500 vines) from theexperimental vineyard of Instituto Superior de Agronomia,located in Tapada da Ajuda, Lisbon, Portugal (latitude 38°42′31.57″ N and longitude 9° 11′14.01″ W), was visuallyinspected just after veráison, in order to detect bunches withinitial symptoms of sour rot. At this phase, two sets of threedifferent vines (duplicates) containing bunches with the firstdisease symptoms were selected and marked. The samenumber of vines containing only healthy bunches were usedas control vine sets. The microbial yeast population presenton both sound and with initial sour rot symptoms buncheswas studied at this grape ripening phase (19 August 2008).

In order to study the effect of Drosophila flies on diseasedevelopment, two types of bunches were studied: (a) freebunches without impediments for the action of any vectorand (b) bunches protected inside a handmade plastic structure(15×15 mm plastic square mesh) covered by a transparent

commercial nylon cloth with a pore size sufficiently small toprevent the entrance ofDrosophila flies and other insects. Theplastic net was adjusted to the size of each selected bunch andthe nylon cloth assured air circulation and sunlight entry. Atotal of six different experimental modalities were analyzed:(a) free sound bunches (SF); (b) protected sound bunches (SP);(c) free sound bunches with induced wounds (SwF) using asterile scalpel blade; (d) protected sound bunches with in-duced wounds (SwP); (e) free bunches with sour rot symptoms(SRF) and (f) protected bunches with sour rot symptoms (SRP)(Fig. 1). The selected bunches were left in the vineyard duringthe ripening period and subsequently collected at the time ofharvest (11 September 2008) and used for the analyses of themicrobial yeast flora and chemical composition of their re-spective grape musts.

Microbial Populations of Yeasts from Grape Samples

Just after the veráison, a total of three bunches (one pervine) of both sound and affected grapes were collected fromeach vine set. The same grape sampling was used for thebefore mentioned six trial modalities, analyzed at the harvesttime. Duplicates were performed for all grape samples.Bunches were collected from vines and transported to thelaboratory in sterile bags.

Representative grape berries were carefully removedfrom each set of three bunches and combined to give a totalof 300 g sample. Berries were then placed in a sterilestomacher bag and crushed in a stomacher blender (IUL,Barcelona, Spain) during 5 min, and the obtained grapejuices were subsequently serially diluted (10−1 to 10−6) inpeptone water (Merck, Darmstadt, Germany).

The yeast flora recovered from grape musts was analyzedby plating (0.1 mL) the diluted grape juice onto the follow-ing media: (a) general purpose medium GYP agar [17] and(b) selective/differential media DBDM [23] and ZDM [24]for the isolation of Dekkera/Brettanomyces and Zygosac-charomyces species, respectively, or other non-Saccharomy-ces species as previously reported [18, 19]. Plates wereincubated at 25°C up to 5 days in the case of GYP platesand 12 days for DBDM and ZDM media. Total yeast countswere obtained from the GYP plates and reported aslog CFU g−1.

For each analyzed berry sample, plates with count-able number of yeast colonies (1 to 300) were examinedand the different colony morphological types foundwere registered and differentially counted. Representa-tive isolates of each colony type were isolated andselected for identification. All isolates were streakedand purified on GYP plates. For long-term storage,purified isolates were maintained frozen at −80°C invials containing the GYP broth with 15 % (v/v) glycerol(Merck), until their later analysis.

The Role of Drosophila sp. on Sour Rot 417

Capture of Wild Drosophila Flies in the Vineyard and YeastFlora Analysis

One week before harvest, fruit-fly traps were placed in thevineyard surrounding bunches with sour rot in the selectedinfected vines. Berries with sour rot were used as bait andplaced in the bottom of small uncapped 330-mL plasticbottles, placed suspended in the vine canopy. The lowerand upper sections of the bottles were separated by a finenylon cloth layer, used as a physical barrier in order to avoidthe contact between flies and the rotten grapes. Once insidethe traps, flies remained captured after capping the upperpart of bottles. Traps were transported to the laboratory andflies were killed by freezing.

In order to analyze the yeasts carried predominantly onthe external surface of flies, sets of ten intact Drosophilabodies were directly transferred to flasks containing 50 mLof GYP and DBDM enrichment broth (duplicate). Addition-ally, other sets of ten flies were surface sterilized by immer-sion in ethanol 70 % for 1 min and then transferred to sterilePetri dishes lid containing 2 mL of sterile peptone water.Flies were dissected with the aid of a sterile scalpel blade, toanalyze and isolate the yeast species from their digestive

tract. The obtained macerated mixtures were transferred forflasks with 50 mL GYP and DBDM broths. Flasks wereincubated at 25°C in an orbital shaker (Agitorb 200IC,Aralab, Lisbon, Portugal) at 120 rpm up to 5 and 12 daysin case of GYP and DBDM broths, respectively. Afterenrichment, yeast isolates were obtained by streaking analiquot (10 μL) of all media with visible growth in GYPagar plates incubated at 25°C up to 5 days. Representativeisolates of each colony type found were purified by streak-ing on GYP plates and stored at −80°C until identification.

Chemical Analysis of Grape Musts at Harvest Time

Grape must samples of the six studied modalities wereanalyzed by Fourier transform infrared (FTIR) Spectroscopyin a WineScan FT120 spectrometer (FOSS, Hilleroed, Den-mark, http://www.foss.dk) using the FOSS-supplied Grape-Scan Calibration [17, 25]. The following oenological andsanitary parameters were determined: glucose-fructose con-tent, total acidity, pH, tartaric acid, malic acid, volatileacidity and gray rot, sour rot, yeast activity and lactic bac-teria indexes. The results of the sanitary indexes were basedon the following ranges: gray rot index (0–60; 00excellent

Figure 1 Representative samples of free (I) and protected (II) grapebunches used in the vineyard experimental trial. Free bunches underinfluence of insects: sound (SF); sound with manually induced wounds

(SwF) and with initial sour rot symptoms (SRF). Bunches protectedfrom vectors influence: sound (SP); sound with manually inducedwounds (SwP) and initial sour rot symptoms (SRP)

418 A. Barata et al.

grapes; 100good grapes; 200slight attack; 300critical at-tack; 400serious attack; 500severe attack; 600 total rottengrapes), sour rot index (0–30; 00good grapes; 100slightattack; 200serious attack; 300severe attack), yeast activityindex (0–30; 00good grapes; 100slight attack; 200seriousattack; 300severe attack), and lactic bacteria activity index(0–2; 00good grapes, 10medium attack, 20severe attack).

In-Vitro Sour Rot Development Tests

A laboratory method was developed in order to study the roleof the fruit fly Drosophila sp. on development of sour rotgrape disease. Commercial white table grapes cv. Italia andlaboratory-rearedDrosophila sp. were used in the in vitro sourrot assays. Healthy grape berries (with pedicel in place) wereremoved from the bunches and inoculated separately withyeast strains belonging to ten different species, one AABspecies and one lactic acid bacteria species, after a previousphase of rinsing, surface disinfection, and washing. The de-tailed procedure is shown in Figure S1 (see Supplementarymaterial). With the exception for Saccharomyces cerevisiaeISA 1000 strain [26], used as control yeast, the remainingselected strains belong to the typical species consortium

isolated from the surface of sour rotten grapes or rotten grapemusts which have been isolated in our laboratory in previousstudies [17–19]. The inoculated strains and their origins arelisted in Table S4 (see Supplementary material).

After surface sterilization and rinsing in sterile distilledwater, each berry set was immersed in a yeast or bacterialcellular suspension (107 cells mL−1) prepared in 300 mL ofsterile peptone water. The inoculum of each tested strainwas prepared from a loopful of fresh culture (24–48 h)inoculated in 50 mL GYP culture broth and incubated at25°C with orbital shaking (120 rpm). Growth was followedby measurement of the absorbance at 640 nm and when ODwas about 1.0 unit, the appropriate inoculum volume wascentrifuged (10,000 rpm, 5 min), cells were washed twicewith peptone water and finally resuspended in 300 mL ofpeptone water.

For each inoculated strain, four types of grape sampleswere analyzed: (a) sound berries (control set) (C); (b) soundberries in the presence of Drosophila sp. flies (D); (c)wounded berries (W) and (d) wounded berries in the pres-ence of Drosophila sp. flies (WD) (Fig. 2). Wounds wereperformed prior to inoculation using a sterile scalpel blade.After inoculation, grape berries sets were placed inside a

Figure 2 Example of the four grape modalities used in the in-vitrosour rot development assay. Inoculated strain: S. cerevisiae yeast strainISA 1000. Modalities: C (control set) sound berries, D sound berries inthe presence of Drosophila sp. flies; W wounded sound berries; WD

wounded berries in the presence of Drosophila sp. flies. Stages: I justafter strain inoculation, II incubation in sterile 1 L cup during 12 days,III final berries sanitary state after incubation

The Role of Drosophila sp. on Sour Rot 419

sterile 1-L glass cup and in case of sets D and WD, tenreared adult Drosophila flies (five males and five females)previously anesthetized were placed inside the cups. Finally,cups were sealed with nylon cloth. Evaluation of the role ofDrosophila was based on sour rot symptoms development(berries browning, dehydration and disaggregation of theinternal tissues and release of a pungent odor of acetic acid)after 12 days of incubation at 25°C in a climate-controlledroom.

Laboratory-Reared Drosophila sp. Fly Stocks and GrowthConditions

The Drosophila flies used in the in vitro sour rot develop-ment assays were reared from initial wild Drosophila sp.adult flies captured during the summer of 2009. Several fly-traps similar to those used in the previously described 2008experimental trial performed on the vineyard (330-mL plas-tic bottles) were placed in an well ventilated room of ourlaboratory. Inside the traps, rotten grapes with a strongvinegar odor were used as bait to attract the flies. Onceinside the traps, flies were anesthetized with ether andtransferred to 23×75 mm sterile disposable polystyrenevials (Sarstedt, Nümbrecht, Germany) with cotton closure,containing nutrient Drosophila medium (NDM) composedby 20 gL−1 yeast extract (Oxoid, Hampshire, UK), 70 gL−1

commercial corn flour, 75 gL−1 sucrose (JTBaker, Deventer,Holland), 45 gL−1 malt extract (Oxoid) and 10 gL−1 agar,supplemented with 25 mL L−1 of the antiseptic Nipagin-M(José M. Vaz Pereira, S.A., Benavente, Portugal) 10 % inethanol 96 % solution (methyl 4-hydroxybenzoate).

Stock flies were reared and maintained for several gen-erations on NDM vials (10 pairs/vial) incubated at roomtemperature (20–25°C) in the laboratory.

Enumeration and Isolation of Yeasts and BacteriaDuring In Vitro Assays

The microbial populations of yeasts and bacteria present ongrape surfaces were determined initially (just after inocula-tion of each tested strain) and after 12 days of berry incu-bation, whenever sour rot development was observed.Berries were crushed in a sterile stomacher bag and grapemusts were analyzed by surface plating (0.1 mL) of theadequate dilutions onto GYP agar plates supplemented with100 mg L−1 chloramphenicol (Sigma, Steinheim, Germany)and TSA plates (Oxoid) supplemented with 200 mg L−1

Delvocid® (DSM, Delft, Netherlands; 100 mg L−1 natamy-cin), for the enumeration of total yeasts and bacteria, respec-tively (see Figure S1). Plates were incubated up to 5 days at25 and 28°C for GYP and TSA plates, respectively.

For the grape sets inoculated with Enterococcus duransstrain ISA 4315, initial and final bacterial enumeration were

carried out using MRS plates [52 gL−1 MRS (Oxoid),200 mg L−1 Delvocid®, 0.5 gL−1 L−cysteine (Sigma),20 gL−1 agar].

The countable plates used in the final microbial analysiswere visually analyzed. Colonies showing different morpho-logical characteristics than those of the inoculated strainwere registered and representative isolates of those colonieswere isolated, purified by streaking on GYP and TSA agarplates, in case of yeasts or bacteria, respectively, and storedat -80°C until identification.

Yeasts and AAB Flora of Drosophila sp. Reared Flies

A total of 50 reared Drosophila sp. stock flies (25 males and25 females) were anesthetized with ether and macerated in5 mL of sterile peptone water using a sterile mortar andpestle.

Afterwards, aliquots of 1 mL of the macerate were trans-ferred to flasks containing 50 mL of GYP supplementedwith 100 mg L−1 chloramphenicol and DBDM broths, bothfor the isolation of yeasts species and GY broth [50 gL−1

glucose, 10 gL−1 yeast extract, and 13 gL−1 agar,200 mg L−1 Delvocid® (DSM, Delft, Netherlands;100 mg L−1 natamycin), 3,000 UL−1 penicillin (Sigma),pH 4.5], used as a specific enrichment media for AAB.Flasks were incubated in an orbital shaker (Aralab) at120 rpm up to 5 and 12 days at 25°C, in the case of GYPand DBDM media, respectively, and 28°C up to 5 days inthe case of GY media. After enrichment, yeast and bacterialisolates were obtained by streaking 10 μL (triplicates) ofeach media with microbial development onto GYP and GYplates, respectively. Representative isolates of each type ofcolony/cell morphology found on plates were isolated, pu-rified and selected for identification.

Acetic Acid and Gluconic Acid Determinations

The two main chemical markers of sour rot were quantifiedfor those grape sets which showed sour rot symptoms at thefinal of in-vitro tests. Acetic acid was determined in the finalgrape musts samples using a Reflectoquant RQflex10®(Merck, Darmstadt, Germany) device and the Reflecto-quant® Acetic Acid test strips (Merck), according to themanufacturer’s instructions. Gluconic acid was quantifiedusing the D-gluconic acid/D-glucono-δ-lactone enzymaticassay kit (Megazyme International Ireland Ltd, Wicklow,Ireland), according to the manufacturer’s instructions.

Identification of Yeasts Isolates

Yeast isolates recovered from grapes and Drosophila sp.flies (2008 vintage) and 2009 in-vitro trials were identifiedby restriction fragment length polymorphism (RFLP)

420 A. Barata et al.

analysis of the 5.8S-ITS rDNA region [27]. PCR amplifica-tions were carried out from fresh colonies (48 h) resus-pended directly in the PCR mixture, or from extractedDNA obtained after a previous enzymatic treatment withliticase (5 mg/mL), following the procedure described inCryer et al. [28]. PCR amplification conditions, restrictionreactions with CfoI, HaeIII and HinfI (Bioron, GmbH, Lud-wigshafen, Germany) restriction endonucleases and electro-phoretic conditions were performed as described in Barata etal. [18]. Restriction patterns were determined by comparisonwith 100-bp DNA ladder (Bioron) using the Quantity One1-D analysis software (BioRad) and compared with thosecontained in the Yeast-id database at www.yeast-id.com(Valencia University and CSIC, Spain).

Yeast isolates sharing similar restriction patterns or withmisidentified patterns, were grouped and representative iso-lates of each case were identified by sequencing the D1/D2domains of the 26S rRNA gene as described in Barata et al.[18]. Sequences were edited and assembled using BioEditSequence Alignment Editor version 7.0.1 software [29], andthen subjected to the GenBank BLASTN search tool of theNCBI database (http://ncbi.nlm.nih.gov/blast) to retrievesequences of closely related taxa.

The cluster Hanseniaspora uvarum/Hanseniaspora guil-liermondii/Hanseniaspora opuntiae was differentiated bygrowth on GYP agar plates incubated at 37°C [30] and byrestriction of 5.8S-ITS region with DdeI and DraI (Bioron)endonucleases [31, 32]. The Lachancea clade former spe-cies (Lachancea fermentati, Lachancea cidri, Lachanceathermotolerans, and Lachancea waltii) were differentiatedby growth on GYP agar plates incubated at 37°C and 40°C,and growth with D-galactose and 0.01 % cycloheximide[33, 34]. Candida stellata was distinguished from Candidazemplinina by digestion of the 5.8S-ITS region with DraI(Bioron) endonuclease [35].

Identification of AAB Isolates

Gram-negative, catalase positive isolates with ellipsoidal torod-shaped cells were considered as putative acetic acidbacteria. These isolates were identified by RFLP of PCR-amplified 16S rRNA gene and RFLP of PCR-amplified16S-23S ITS region according to González et al. [36].DNA extraction was carried out as follows: fresh cultureswere grown on GYplates and three to five bacterial colonieswere picked and ressuspended in 50 μL of lysis solution(0.25 % sodium dodecyl sulfate, 0.05 N NaOH) and incu-bated at 100°C for 15 min (boiling water bath). The suspen-sion was cooled at room temperature, centrifuged (5,000×g,4°C, 5 min) and the supernatant containing the releasedDNA was diluted 100-fold with sterile milliQ water andused as template for PCR amplification. The 16S rRNAgene and 16S-23S ITS regions were amplified using the

primers and thermocycling conditions designed by Ruiz etal. [37]. PCR amplifications were carried out in 50 μLreaction volume, consisting of 2 μL bacterial DNA lysateand 48 μL amplification mixture, containing 10 μL of 5×amplification buffer Green Go Taq Flexi (Promega),200 μM of dNTPs mixture, 2 mM MgCl2, 0.3 μM of eachprimer and 2.5 U Go Taq® (Promega). For the restrictionreactions of the 16S rRNA gene-amplified products, 5 μL ofeach PCR product was digested with 10 U of AluI (Fermen-tas, Thermo Fisher Scientific Inc., Leicestershire, UK), CfoI,HinfI, Tru9I (Bioron) and TaqI (Metabion, Martinsried,Germany) restriction endonucleases, as recommended bythe manufacturers.

Isolates inconclusively identified by RFLP of 16S rRNAgene Acetobacter cerevisae/Acetobacter orleaniensis/Aceto-bacter malorum, were resolved by RFLP of 16S–23S ITSregion, digesting the amplified 16S–23S ITS product (5 μL)with AluI and CfoI endonucleases [36].

The PCR products and restriction fragments weredetected and separated by 1.0 % (w/v) and 3.5 % (w/v)agarose gel electrophoresis in 1× TBE buffer, respectively.Restriction patterns were obtained after estimation of frag-ment sizes by comparing their mobility against a 100-bpDNA Ladder (Bioron), as described above.

Results of RFLP analysis were confirmed by sequencingthe amplified 16S rRNA gene of representative isolates ofeach restriction pattern. For those isolates, the amplified 16SrDNA products were purified, sequenced, and the finalcorrected sequences were submitted to the BLASTN net-work service, as previously described.

Nucleotide Sequences Accession Numbers

Nucleotide sequences were deposited in the NCBI GenBankdatabase library under the accession numbers JN004183 toJN004201.

Results and Discussion

Effect of the Grape Cluster Coverage on the Developmentof Sour Rot

The results listed in Table 1 showed that the development ofsour rot could be prevented if wounded berries were pro-tected from the contact of insects during ripening. Asexpected, healthy bunches with unblemished berries main-tained their perfect sanitary state until harvest, independent-ly of the presence of vectors. When sound bunches werewounded and left without protection (SwF), two differentresults were observed: for 50 % of the cases the woundedberries rotted, while the other 50 % showed healing of theskin. On the contrary, when wounded bunches were

The Role of Drosophila sp. on Sour Rot 421

protected inside the nylon nets (SwP), all of them healed andthus did not show any sign of rotting until harvest.

Concerning grape samples with sour rot symptoms pro-tected with nylon nets (SRP), results showed that in mostbunches the spread of sour rot for the remaining parts of theclusters was avoided. Nevertheless, one bunch in each vineset rotted completely. In this case, we observed adult Dro-sophila spp. flies and larvae at the harvest time inside thenets, which possibly hatched from eggs or larvae alreadypresent on the initially affected berries.

Finally, the six grape bunches (vine set A and B) withinitial signs of sour rot and left free during ripening (SRF),were totally rotten at the harvest time, emphasizing the roleof Drosophila spp. for the development and disease exten-sion. Moreover, the constant presence of Drosophila fliessurrounding the rotten clusters and a large number of larvaeinside the rotten berries were observed.

Effect of Drosophila spp. on Yeast Populations Isolatedfrom Grapes

A total of 159 yeast strains representative of the diversityfound in all analyzed grape samples were isolated andselected for identification. The RFLP of 5.8S-ITS analysisperformed for these isolates generated 26 different restric-tion patterns obtained with CfoI, HaeIII, Hinf, DdeI andDraI restriction endonucleases, of which 11 were success-fully identified by comparison with the restriction profilescontained in the Yeast-id database, while 15 were unidenti-fied by this method. Representative isolates of each patternincluded in the latter group were subsequently identified bysequencing the D1/D2 domains of the 26S rRNA gene.Detailed restriction fragments data of each identified yeastspecies by PCR-RFLP of the 5.8S-ITS region are availableas supplementary material (Table S4).

Table 2 shows the diversity and enumeration of yeastsspecies recovered from the six grape modalities analyzed at

harvest time after being left protected or unprotected fromvectors during ripening. Just after veráison, as previouslyobserved [18], the amount and diversity of yeasts specieswas significantly changed on bunches with initial sour rotsymptoms. Sour rotten bunches had on average more4.25 log CFU g−1 of total yeast counts than the healthygrapes samples, and showed 13 different ascomycetousyeast species, dominated by C. zemplinina, C. apicola, andH. uvarum, while basidiomycetes were not detected. More-over, sour rotten samples showed the presence of the sourrot zymological markers Zygoascus hellenicus, Pichia terri-cola, and Pichia kudriavzevii (the two latter species wereformerly named as Issatchenkia spp.), in accordance withour previous studies [18, 19]. On the contrary, sound grapeshad lower yeasts species diversity characterized by threeascomycetous (Candida diversa, C. zemplinina, and H. uva-rum), three basidiomycetous yeast species (Cryptococcusmagnus, Cryptococcus carnescens, and Rhodotorula spp.)and by the dominance of the ascomycetous yeast-like fungusAureobasidium pullulans.

All protected and free sound bunches (SP and SF)remained healthy until the harvest time (see Table 1) andsimilar species diversity was found on both sound bunchesmodalities in comparison with the initial ripening phase.Since veráison, yeast flora was characterized by severalbasidiomycetous species (Cryptococcus flavescens, Crypto-coccus laurentii, Sporobolomyces roseus, and Rhodotorulaspp.) and by the dominance of the ascomycetous species C.zemplinina and A. pullulans (Table 2). These results showedthat unblemished berries bore constant numbers and speciesdiversity through ripening and have similar variability tothat found on the surface of other parts of the phylloplane[38].

Grape microbiota of sound berries artificially woundedwas changed according to the protection from insect contact.In case of protected bunches (SwP), species diversity wassimilar to that of mature healthy bunches (see Table 2),

Table 1 Effect of shielding grape bunches with nylon nets on sour rot development. Results reported as the number of bunches associated witheach sanitary state

Assay Sanitary state at veráison Bunches Sanitary state at harvesta

Sound Wound’s healing Partially rotten Totally rotten

(A) (B) (A) (B) (A) (B) (A) (B)

SF Sound Free 3 3

SP Protected 3 3

SwF Sound with wounds Free 2 1 1 2

SwP Protected 3 3

SRF Sour rot symptoms Free 3 3

SRP Protected 2 2 1 1

a (A) and (B) represent the two independent sampling (vine sets)

422 A. Barata et al.

Tab

le2

Microbialyeastpo

pulatio

nsisolated

from

soun

dbu

nchesandwith

initialsour

rotsymptom

safterveráison

andeffectof

bunchesshieldingdu

ring

ripening

onyeastdy

namicpo

pulatio

ns

Yeastspeciesa

After

veráison

bHarvesttim

ebGenBankAcessionno

.c

Protected

buncheswith

net

Freebu

nches

SSR

S PSwP

SRP

S FSwF

SRF

(A)

(B)

(A)

(B)

(A)

(B)

(A)

(B)

(A)

(B)

(A)

(B)

(A)

(B)

(A)

(B)

Ba

Cryptococcusau

reus

3.40

JN00

4192

Cryptococcuscarnescens

1.18

JN00

4185

Cryptococcusfla

vescens

0.70

1.78

JN00

4195

Cryptococcuslaurentii

2.74

Cryptococcusmag

nus

3.18

JN00

4184

Cryptococcusspp.

3.00

2.70

3.00

JN00

4190

Sporob

olom

yces

roseus

0.70

3.70

3.70

2.48

2.88

Rho

dotorula

spp.

2.70

1.00

1.00

2.70

JN00

4191

As

Aureobasidium

pullu

lans

3.30

2.08

4.07

3.89

3.30

3.54

4.90

4.48

4.40

4.18

3.54

3.54

4.18

5.98

5.00

4.70

JN00

4193

Aureobasidium

spp.

1.70

JN00

4186

Can

dida

amap

ae2.18

1.18

1.65

1.48

Can

dida

apicola

6.29

6.28

6.39

6.93

7.73

7.11

JN00

4197

Can

dida

azym

a4.24

2.95

4.13

5.23

JN00

4198

Can

dida

diversa

3.00

Can

dida

zemplinina

2.48

7.08

7.23

4.06

2.60

3.08

3.40

5.78

6.49

4.04

3.81

4.48

6.39

6.00

7.11

JN00

4183

Han

seniaspo

ragu

illierm

ondii

5.42

5.28

3.48

5.65

5.44

2.95

3.18

3.30

6.09

6.07

Han

seniaspo

rauvarum

1.60

6.12

5.98

3.81

2.93

5.81

5.00

2.74

3.30

6.48

6.60

JN00

4196

Lachancea

thermotolerans

5.65

4.70

4.18

4.70

5.00

Metchnikowia

pulcherrima

4.18

5.22

4.74

3.12

4.25

4.18

3.40

Pichiakluyveri

3.00

5.13

JN00

4187

Pichiakudriavzevii

5.40

6.00

JN00

4188

Pichiasporocuriosa

3.52

2.18

2.26

2.30

JN00

4199

Pichiaterricola

5.78

5.40

5.30

4.70

3.87

5.35

5.48

5.48

Saccharomycop

sisvini

5.00

4.93

1.90

3.20

3.74

5.00

Zygoa

scus

hellenicus

4.90

4.14

5.24

4.12

4.05

4.92

5.73

5.23

Zygosaccharom

yces

bisporus

4.70

5.88

4.63

6.00

6.30

Totalcounts

3.72

2.29

7.18

7.31

4.22

3.69

5.01

4.59

6.62

7.10

4.18

4.12

4.75

6.57

7.77

7.51

Resultsrepo

rted

aslogCFU

g−1

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ycetes,Asc

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bTyp

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d,SR

sour

rotsymptom

s,Sw

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nds;(A

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cGenBankaccessionnu

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NCBIGenBankdatabase

library

The Role of Drosophila sp. on Sour Rot 423

which is in accordance with the final sanitary state observedat harvest, characterized by the total healing of their woundsand absence of any type of sour rot symptoms (Table 1). Onthe contrary, wounded bunches visited by Drosophila flies(SwF), were characterized by higher total yeast populations(5.66 CFU g−1 in average of both sampling sets), absence ofbasidiomycetes, and the presence of the typical sour rotcolonizing yeasts P. terricola, Z. hellenicus, and Zygosac-charomyces bisporus (Table 2).

Finally, identical yeast species were recovered from bothcovered (SRP) and free (SRF) bunch sets with initial sour rotsymptoms. Thirteen ascomycetous yeast species were iso-lated from these samples, among which high populations ofZ. bisporus. This similarity in yeast diversity did not reflect,however, the final grape sanitary state of bunches at harvesttime. In fact, free bunches were fully rotten while for mostof the protected bunches sour rot symptoms were restrictedto the early infected berries (Table 1).

Isolation of Yeasts from Wild Drosophila sp. Flies

The enrichment strategy used for the isolation of yeasts fromwild Drosophila spp. flies captured in the vineyard, allowedthe recovery of 20 yeast isolates from the bodies surface(10) and digestive tract of flies (10). Three different RFLPpatterns were obtained in each group of isolates. Thesepatterns matched with the yeast species C. zemplinina, H.uvarum, and Z. hellenicus, for the group of bodies surfaceisolates and H. uvarum, P. terricola, and Z. hellenicus, incase of isolates obtained from the Drosophilamacerated sets(Table 3).

These results are in accordance with previous studies [22,39], which showed by electron microscopy the presenceKloeckera apiculata (anamorph of H. uvarum) yeasts cellson the bodies surface (thorax and legs) of adult Drosophilaflies. These authors also analyzed the microbiota of thesurface and gut of adult flies captured in vineyards of threedifferent regions in France and isolated K. apiculata, C.stellata and M. pulcherrima either from the flies’ surfaceor gut, and also P. membranifacies and C. krusei only fromthe gut.

The consistent isolation of P. terricola and Z. hellenicus,only from sour rotten grapes is in accordance with ourprevious studies [18, 19]. Therefore, these results indicateclearly that Drosophila spp. flies are vectors of the mainyeast flora that typically colonize the surface of sour rottengrapes and support the empirical hypothesis which holdsthat fruit flies are involved in the spread of grapevine sourrot.

Influence of Bunch Protection on Chemical Compositionof Grape Musts

Table S2 (see Supplementary material) illustrates the effectof bunch protection on the changing of the oenologicalparameters of grape musts. The results obtained by FTIRanalysis showed that several chemical parameters were sig-nificantly affected when wounded sound (Sw) or with initialsymptoms of sour rot (SR) bunches were kept protectedfrom the influence of insects. In order to evaluate the sig-nificance of these changes, a set of unpaired t tests wasperformed between must samples obtained from protectedand free bunches. Table S3 (see Supplementary material)summarizes the oenological parameters with significant dif-ferences between samples. The samples of grape mustsobtained from wounded sound bunches visited by Drosoph-ila flies, showed significantly higher values of total acidity,volatile acidity, accompanied with a significant drop of pH,when compared with those obtained from the coveredbunches. Moreover the results of the sour rot and yeastactivity sanitary indexes were significantly higher for thefree bunch samples, which were classified as having a“slight attack” contrasting with the classification of “goodgrapes” for the covered bunch.

Indeed, these results are in agreement with the previouslydescribed microbial observations of the final sanitary char-acteristics of bunches at harvest (Table 1).

Interestingly, the chemical profile of the grape must sam-ples obtained from the covered wounded bunches (SwP),was similar to those registered for both sets of soundbunches (SF and SP). Bearing in mind that the totality ofwounded bunches which were not visited by Drosophila

Table 3 Yeast species recovered from the surface and macerate of wild Drosophila sp. flies captured in the vineyard

Isolate RFLP Pattern Yeast species Captured wild Drosophila sp.

Intact Macerated

GYP DBDM GYP DBDM

WD1, WD2, WD3 Y15 C. zemplinina + − − −

WD4, WD5,WD11, WD12, WD13 Y18 H. uvarum + − + −

WD14, WD15 Y25 P. terricola − − + −

WD6, WD7, WD8, WD9, WD10, WD16, WD17,WD18, WD19, WD20 Y28 Z. hellenicus + + + +

424 A. Barata et al.

Table 4 Effects of the microbial species and of Drosophila sp. during the development of sour rot in-vitro

Inoculated strain Assaya Initial countsb

(log CFU g−1)Berries characterizationc Final countsb

(log CFU g−1)Metabolitesd

TY TB 1. 2. 3. 4. Sour rot (%) TY TB Acet (g L−1) Gluc (g L−1)

C. zemplinina C 5.56 <1e − − − n.a. 0 n.d. n.d. n.d. n.d.

ISA 2339 D 5.35 <1 − − − n.a. 0 n.d. n.d. n.d. n.d.

W 5.47 <1 + − − − 0 7.93 <1 0.24 0.07

WD 5.29 <1 ++ ++ ++ − 86 7.55 6.81 7.00 10.79

H.uvarum C 5.06 <1 − − − n.a. 0 n.d. n.d. n.d. n.d.

ISA 2294 D 5.08 <1 − − − n.a. 0 n.d. n.d. n.d. n.d.

W 5.15 <1 + − − + 0 7.25 <1 0.11 0.01

WD 5.19 <1 + ++ ++ − 71 6.70 7.38 3.37 0.98

I. occidentalis C 4.60 <1 − − − n.a. 0 n.d. n.d. n.d. n.d.

ISA 2384 D 4.68 <1 − − − n.a. 0 n.d. n.d. n.d. n.d.

W 5.06 <1 + − − − 0 8.11 <1 0.10 0.09

WD 5.13 <1 ++ ++ ++ − 71 8.29 6.78 1.73 1.22

I. orientalis C 5.75 <1 − − − n.a. 0 n.d. n.d. n.d. n.d.

ISA 2381 D 5.53 <1 − − − n.a. 0 n.d. n.d. n.d. n.d.

W 5.53 <1 + − − + 0 6.92 <1 0.08 0.07

WD 5.38 <1 ++ ++ + − 100 7.72 5.48 0.75 0.09

I. terricola C 4.53 <1 − − − n.a. 0 n.d. n.d. n.d. n.d.

ISA 2322 D 4.41 <1 − − − n.a. 0 n.d. n.d. n.d. n.d.

W 4.70 <1 + − − + 0 6.38 <1 0.09 0.06

WD 4.66 <1 + + + − 86 6.64 4.78 0.58 0.17

L. thermotolerans C 5.38 <1 − − − n.a. 0 n.d. n.d. n.d. n.d.

ISA 2380 D 5.60 <1 − − − n.a. 0 n.d. n.d. n.d. n.d.

W 5.48 <1 + − − − 0 7.70 <1 0.14 0.01

WD 5.53 <1 + ++ ++ − 86 6.87 7.26 4.50 0.51

S. cerevisiae C 4.89 <1 − − − n.a. 0 n.d. n.d. n.d. n.d.

ISA 1000 D 4.56 <1 − − − n.a. 0 n.d. n.d. n.d. n.d.

W 5.41 <1 + − − + 0 7.34 <1 <0.04 0.01

WD 5.45 <1 ++ +++ +++ − 100 7.19 >8.60 3.67 8.73

Z. hellenicus C 5.34 <1 − − − n.a. 0 n.d. n.d. n.d. n.d.

ISA 2324 D 5.02 <1 − − − n.a. 0 n.d. n.d. n.d. n.d.

W 4.53 <1 + − − − 0 8.44 <1 0.25 0.04

WD 5.35 <1 ++ ++ ++ − 71 7.58 5.56 1.19 7.07

Z.bailii C 5.16 <1 − − − n.a. 0 n.d. n.d. n.d. n.d.

ISA 2295 D 5.18 <1 − − − n.a. 0 n.d. n.d. n.d. n.d.

W 5.51 <1 + − − − 0 7.51 <1 0.04 0.08

WD 5.34 <1 ++ +++ +++ − 100 6.53 5.00 9.20 2.49

Z. bisporus C 5.30 <1 − − − n.a. 0 n.d. n.d. n.d. n.d.

ISA2336 D 5.26 <1 − − − n.a. 0 n.d. n.d. n.d. n.d.

W 5.56 <1 + − − − 0 8.00 <1 0.10 0.10

WD 5.68 <1 ++ ++ + − 100 7.97 <1 0.49 0.17

Ga. saccharivorans C <1 5.73 − − − n.a. 0 n.d. n.d. n.d. n.d.

CBISA 4244 D <1 5.62 + + ++ n.a. 28 6.87 6.00 2.05 7.15

W <1 5.58 + ++ ++ − 71 <1 6.98 1.43 8.02

WD <1 5.79 +++ +++ +++ − 100 7.64 6.21 12.80 29.20

E. durans C <1 4.72 − − − n.a. 0 n.d. n.d. <0.04 n.d.

CBISA 4315 D <1 5.06 − − − n.a. 0 n.d. n.d. <0.04 n.d.

W <1 5.15 + − − + 0 n.d. n.d. <0.04 n.d.

WD <1 4.72 + +++ + − 100 8.39 7.83 0.41 5.56

The Role of Drosophila sp. on Sour Rot 425

flies, or other insects, healed their wounds, we speculate thatthe vine plant activated a defense mechanism leading to thehealing of berry skin lesions.

A similar trend was observed for the free and protectedbunches with initial sour rot symptoms. Table S3 shows thatthe musts from free bunches were characterized by signifi-cant higher values of glucose-fructose, total acidity, volatileacidity, sour rot index, and a slight lowering of pH values,than those kept in the absence of the vectors. These chem-ical changes were recently defined as the typical changingpattern arising from the sour rotting process [17]. The vec-tors role became clear since significant lower levels ofvolatile acidity and sour rot index were observed in thecovered bunches. These bunches were classified as “slightattack” in contrast to the “serious to severe attack” associ-ated to free bunches. Therefore these results clearly suggestthat Drosophila flies could be the main sources of AABwhich typically colonize the surface of sour rotten grapes, asstated by [39].

No significant differences were found between both pro-tected and free sound bunches for all the parameters tested,which shows that Drosophila flies induced chemicalchanges in musts only when the physical barrier posed bythe berry skin was broken.

In Vitro Sour Rot Development Tests

The developed method involved the use of commercial tablegrapes inoculated with several strains of yeasts and bacteriaspecies belonging to the typical microbiota of sour rottenberries, after sequential steps of rinsing, surface disinfectionand washing of the berries. After microbial inoculation,healthy and wounded berries were incubated with the pres-ence and absence of adult Drosophila sp. flies, which were

reared in the laboratory from wild capture Drosophila flies.Thus, it was possible to create in the laboratory, an in vitromethod able to induce the typical symptoms of sour rot andfind the biological agent without which sour rot does notoccur. Table 4 summarizes the data obtained during the invitro sour rotting assays.

The results of the initial microbiological analyses whichwere carried out just after berry inoculation with each indi-vidual tested yeast or bacteria strain, showed that the berrieswere efficiently disinfected, since only the inoculated strainwere recovered. The immersion of berries in a cell suspen-sion containing 107 cells/mL of each selected strain allowedthe production of berries with an average initial microbialcharge of 5.19 and 5.30 log CFU g−1, in case of yeasts andbacterial strains, respectively.

Concerning yeast inoculation trials, Table 4 shows thatindependently of the inoculated strain, healthy and unblem-ished berries either in the absence (C) or presence (D) ofDrosophila flies did not develop any symptoms of sour rot.In all of these trials, none of the sour rot symptoms wereobserved, whereby no further microbial and chemical anal-yses were performed.

Interestingly, the induction of wounds in sound berriesfollowed by the inoculation with the yeasts strains, did notcontribute by itself for the developing of sour rot. Indeed,none of the tested yeast strain induced the disease. More-over, wounds healed for 40 % of the tested yeast strains (H.uvarum, P. kudriavzevii, P. terricola, and S. cerevisiae). Thisphenomenon was previously observed in vivo during theexperimental survey performed on the vineyard, in all thetested wounded sound bunches which were not visited bythe vectors and also in 50 % of the unprotected woundedbunches (Table 1). The final microbiological analyses car-ried out after the in-vitro incubation period, showed that all

Table 4 (continued)

Inoculated strain Assaya Initial countsb

(logCFUg−1)Berries characterizationc Final countsb

(logCFUg−1)Metabolitesd

TY TB 1. 2. 3. 4. Sour rot (%) TY TB Acet (gL−1) Gluc (gL−1)

Control berries Blank 2.64 1.90 n.d. n.d. n.d. n.a. n.d. n.d. n.d. n.d. n.d.

(n.i.) C <1 <1 − − − n.a. 0 n.d. n.d. <0.04 0.03

D <1 <1 − − − n.a. 0 n.d. n.d. 0.16 0.16

W <1 <1 − − − + 0 n.d. n.d. 0.20 0.08

WD <1 <1 + + + − 43 4.08 5.51 0.47 6.06

n.a. not applicable; n.d. not determined; n.i. not inoculatedaC sound berries (control set), D sound berries with Drosophila flies,W sound wounded berries,WD sound wounded berries with Drosophila flies,Blank unsterilized sound berriesb TY total yeasts (GYP plates), TB total bacteria (TSA plates for yeasts and control berries trials, MRS plates for LAB trial)c 1 Browning, 2 disaggregation of the internal tissues, 3 acetic acid odor, 4 wounds healingdAcet acetic acid, Gluc gluconic acide <1 CFU g−1

426 A. Barata et al.

yeasts strains increased their populations at the surface ofwounded berries (2.31 log CFU g−1 in average), and noviable bacterial cells were found (<1 CFU g−1). In addition,low concentrations of acetic acid and gluconic acid weredetected, independently of the yeast strains inoculated (Ta-ble 4). These results seem to demonstrate that none of the

yeast species had the ability to induce sour rot by itself andshould not be regarded as primary agents responsible forthis disease.

A completely different scenario was observed whensound wounded berries were inoculated with the AAB strainof Gluconacetobacter saccharivorans. After incubation,

Table 5 Microbiota recovered from berries sets which developed sour rot during the in-vitro tests

Inoculated species (strain) Assaya Isolates after in-vitro rotting development GenBank accession no.d

Isolateb Colonyc RFLP Pattern Species

C. zemplinina (ISA 2339) WD Lf1 Y + Y15 C. zemplinina

Lf2 Y − Y18 H. uvarum

Bf1 B n.a. B3 G. oxydans

H.uvarum WD Lf5 Y + Y18 H. uvarum

(ISA 2294) Bf6 B n.a. B2 A. malorum

I. occidentalisd WD Lf7 Y + Y23 P. occidentalis

(ISA 2384) Bf3 B n.a. B3 G. oxydans

I. orientalise WD Lf9 Y + Y22 P. kudriavzevii

(ISA 2381) Bf4 B n.a. B2 A. malorum

I. terricolaf WD Lf11 Y + Y25 P. terricola

(ISA 2322) Bf5 B n.a. B2 A. malorum

L. thermotolerans WD Lf25 Y + Y19 L. thermotolerans

(ISA 2380) Bf17 B n.a. B2 A. malorum

S. cerevisiae WD Lf13 Y + Y26 S. cerevisiae

(ISA 1000) Bf7 B n.a. B3 G. oxydans

Z. hellenicus WD Lf3 Y + Y28 Z. hellenicus

(ISA 2324) Bf2 B n.a. B3 G. oxydans JN004204

Z.bailii WD Lf20 Y + Y29 Z. bailii

(ISA 2295) Bf13 B n.a. B3 G. oxydans

Z. bisporus WD Lf18 Y + Y30 Z. bisporus

(ISA2336)

Ga. saccharivorans D Lf16 Y n.a. Y18 H. uvarum

(CBISA 4244) Bf10 B + B4 Ga. saccharivorans

Bf11 B − B2 A. malorum

W Bf12 B + B4 Ga. saccharivorans

WD Lf14; Lf15 Y n.a. Y18 H. uvarum

Bf8 B + B4 Ga. saccharivorans

Bf9 B n.a. B2 A. malorum JN004205

E. durans WD Bf16 B − B1 A. cibinongensis JN004206

(CBISA 4315) Lf23 Y n.a. Y16 C. oleophila JN004207

Control berries WD Lf21 Y n.a. Y15 C. zemplinina

(n.i.) Lf22 Y n.a. Y20 M. pulcherrima

Bf14 B n.a. B3 G. oxydans

Bf15 B n.a. B2 A. malorum

aD sound berries with Drosophila flies, W wounded berries, WD wounded berries with Drosophila fliesb Y yeast, B bacteriac+ colony and cells morphology similar to the inoculated species, − different colony morphologyd GenBank accession number of the sequences deposited in NCBI GenBank database library; n.i. not inoculated; n.a. not applicabled Renamed as Pichia occidentalise Renamed as Pichia kudriavzeviif Renamed as Pichia terricola

The Role of Drosophila sp. on Sour Rot 427

71 % of the inoculated grape berries developed clearly thetypical symptoms of sour rot and high concentration of theacetic (1.43 gL−1) and gluconic (8.02 gL−1) acids werefound in the respective final grape must. These modifica-tions were only due the action of this AAB species, since noviable counts of total yeasts were found after incubation(Table 4).

When inoculated with the AAB strain and maintained withDrosophila adult flies 28 % of the sound berries developedsour rot. However, we believe that this result may have beendue to the presence of small wounds which were not visuallydetected during the selection of the sound berries.

The results obtained with the wounded sound berries setsmaintained in contact with Drosophila spp. (WD), clearlyshowed that Drosophila flies are a key factor without whichno sour rot development occurs. Independent of the yeast orbacterial inoculated strains, all the WD assays culminated inthe development of sour rot and very high concentrations ofacetic and gluconic acids were detected in the respective grapemusts. Regarding yeast inoculation sets, with the exceptionfor the trial inoculated with Z. bisporus, very high bacterialcounts were recovered after incubation of all remaining berriessets, demonstrating that Drosophila flies acts as inoculumsources of bacterial populations responsible for sour rot onset.On the other hand, very high yeasts populations were foundwhen only Ga. saccharivorans (7.64 log CFU g−1) and E.durans (8.39 CFU g−1) were inoculated in wounded berries inthe presence of flies. Moreover, high counts of total yeasts(4.08 CFU g−1) and bacteria (5.51 CFU g−1) were recoveredfrom the wounded control berries (not inoculated) incubatedwith Drosophila. All these results clearly indicate that Dro-sophila flies played a role as inoculation and dispersion vec-tors of the sour rot causal agents.

Identification of Isolates Recovered from In-Vitro SourRotten Berries and Reared Drosophila Flies

Twenty six yeast and sixteen bacterial isolates representativeof the microbial diversity found in all analyzed berries setsafter incubation were selected for identification.

In the case of the wounded berries (W) inoculated with theten different yeasts strains, only a single type of colony mor-phology was found for each individual set, after the incubationperiod. The analysis of the RFLP patterns of the isolates repre-sentative of each morphology confirmed that only the inoculat-ed species were present on the berries surface (data not shown).

Table 5 lists the yeasts and bacterial species isolated fromthe berries sets for which sour rot development was observed.Results showed that beyond the inoculated yeast strain, theyeast speciesH. uvarum and the AAB speciesG. oxydans andA. malorum were recovered after incubation with Drosophilaflies (WD). In particular, G. oxydans and A. malorum wereisolated in 50 % and 40 % of the yeasts inoculation trials,respectively. Furthermore, these two AAB species were alsoisolated from the WD control berries set together with C.zemplinina and M. pulcherrima yeast species.

H. uvarum and A. malorum were also detected in both Dand WD berries set inoculated with Ga. saccharivorans,while Candida oleophila and A. cibinongensis were foundin the WD berries set inoculated with E. durans.

The microbial flora analysis of the reared-Drosophilaflies used in this survey established the origin of most ofthe species found on the in-vitro sour rotten berries.

Six yeast and ten AAB isolates were recovered from amacerate sample of 50 adult reared-Drosophila spp. flies afterthe enrichment step in GYP and GY broths respectively.Among the yeast isolates, two restriction patterns matchedwith the ascomycetous yeasts C. zemplinina and H. uvarum,while for the AAB isolates, G. oxydans, A. malorum and A.cibinongensis were identified (Table 6). As previously ob-served in the analysis of wild captured Drosophila spp. flies,C. zemplinina and H. uvarum were also recovered from thebodies of laboratory-reared flies, which suggest that these twoyeast species could be part of the natural floral of the surfaceor digestive tract of Drosophila flies.

Conclusions

The results of this study together with previous research[40] enabled us to propose a model on the ecological

Table 6 Microbiota isolatedfrom the laboratory-reared Dro-sophila flies

aGenBank accession number ofthe sequence deposited in NCBIGenBank database library

Reared Drosophila flies macerate GenBank Acession noa

Isolate RFLP Pattern Enrichment media Species

L1, L2, L3 Y15 GYP C. zemplinina

L4, L5, L6 Y18 GYP H. uvarum

DR1, DR2, DR3, B3 GY G. oxydans JN004201

DR5, DR8, DR9, DR10 B2 GY A. malorum JN004202

JN004203

DR6 B1 GY A. cibinongensis JF7188429

428 A. Barata et al.

interactions between vine plant, microorganisms andinsects underlying the development of sour rot. In thisvineplant–microorganism–Drosophila model system, thefirst event is the damage of berry skin by any biotic(e.g., grape moths, phytopathogenic molds, birds) orabiotic (e.g., rain, berry abrasion in tight bunches) fac-tors. The injury triggers a plant defense response mech-anism leading to skin healing if Drosophila sp. is notpresent. These insects transport yeasts and acetic acidbacteria that initiate a spontaneous fermentative processon the berry surface. First, fermentative yeasts prolifer-ate and produce ethanol but are not able to induce sourrot alone. Simultaneously, Gluconobacter and Gluconoa-cetobacter spp. also attain high cell numbers, producinggluconic acid and acetic acid. Finally, Acetobacter oxi-dizes ethanol leading to higher concentrations of aceticacid. Therefore, the specific ability to produce aceticand gluconic acid in high levels explain why acetic acidbacteria should be regarded as the etiological agents ofsour rot. The volatiles produced attract larger Drosoph-ila sp. populations spreading sour rot to neighboringinjured berries which physiological defensive responseis not fast enough to heal the skin wounds.

Two main steps on the ecological interaction betweenvine plant, microorganisms and insects need furtherclarification. The first is related with the initial insectattraction signal. Volatile chemicals, such as ethanol,acetic acid, ethyl acetate and acetaldehyde [41, 42],ethyl phenylacetate [43], and acetoin [44] are knownto attract drosophilids. In particular, ethyl phenylacetateand acetoin were found in significantly higher concen-trations in wines affected by sour rot [16]. These latterauthors speculated that the high concentration of phe-nylacetic acid, precursor of ethyl phenylacetate, found insour rotten berries was the result of a plant defensemechanism, because it is a plant auxin [45]. It is con-ceivable that the first event attracting fruit flies is theproduction of phenylacetic acid which acts also as apheromone [45, 46] but we are not aware of specificresearch done with Drosophila sp. The second step isrelated to the mechanisms underlying the prevention ofskin healing in the presence of insects. It is conceivablethat microbial enzymes produced during growth or thefermentative metabolites prevent the curative actionprobably mediated by phenylacetic acid, but furtherresearch is necessary to clarify this issue.

Acknowledgments This work was partially funded by PortugueseScience and Technology Foundation (FCT) and by POCI 2010, partic-ipated by the European fund FEDER under the projects POCI/AGR/56771/2004 and PTDC/AGR-ALI/101393/2008. The authors grateful-ly thank the Analytical Services of Adega Cooperativa de Borbawinery for the grape musts FTIR analyses. A. Barata was the recipientof a PhD grant (Ref. SFRH/BD/28451/2006) from the FCT.

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