REGULAR ARTICLE

Influence of two different cover crops on soil N availability, Nnutritional status, and grape yeast-assimilable N (YAN) in a cv.Tempranillo vineyard

Eva P. Pérez-Álvarez & Teresa Garde-Cerdán &

Pilar Santamaría & Enrique García-Escudero &

Fernando Peregrina

Received: 9 July 2014 /Accepted: 14 January 2015# Springer International Publishing Switzerland 2015

AbstractAim The aim was to study the effects of two differentcover crops (leguminous versus gramineous) on soilNO3

−-N availability, grapevine N status, and yeast-assimilable N (YAN) and amino acid profile in the mustof a cv. Tempranillo vineyard from La Rioja (Spain)over 3 years.Methods Three treatments were established: gramine-ous (barley) and leguminous (Persian clover) covercrops and conventional tillage. Soil total N and extract-able NH4

+-N at bloom, soil NO3−-N content from

budbreak to postharvest, leaf N content at bloom, andfree amino acids and ammonia in must were monitored.Results Each year, the barley cover crop treatmenthad lower soil NO3

−-N than tillage, while theclover treatment showed lower soil NO3

−-N thantillage in 2009 and showed higher values in 2010and 2011. In 2011 leaf N content, yeast-assimilable N (YAN), and some amino acids inthe grapes were lower in barley cover crop treat-ment thus affecting the amino acid profile. Mean-while, the clover cover crop did not have these

effects. Furthermore, relationships between leaf Ncontent and various amino acids were also found.Conclusions With the barley cover crop treatment, soilN availability decreased from the first year of the exper-iment, provoking a decrease in N grapevine status and incontent of YAN and various amino acids at the thirdyear. In contrast, clover cover crop maintained N grape-vine status, YAN, and amino acid profile similar toconventional tillage. Therefore, our study shows howsoil N can be managed both within a year and from yearto year when two different cover crops are sown andhow the grapevine N nutritional status and YAN contentand amino acid profile can be modified in the must.

Keywords Grape amino acids . Leaf nitrogen content .

Soilnitrogenavailability .Mediterraneanvineyard .Winequality

AbbreviationsYAN yeast-assimilable nitrogenAOC appellation d’origine contrôlée

Introduction

Tillage is the common soil management method used inMediterranean vineyards. Various authors have shownthat cover crops decrease the soil N availability in Med-iterranean vineyards (Steenwerth and Belina 2008;Celette et al. 2009; Peregrina et al. 2012; Pérez-Álvarezet al. 2013a). The reduction of soil N availability caused

Plant SoilDOI 10.1007/s11104-015-2387-7

Responsible Editor: Euan K. James.

E. P. Pérez-Álvarez (*) : T. Garde-Cerdán : P. Santamaría :E. García-Escudero : F. PeregrinaInstituto de Ciencias de la Vid y del Vino, ICVV (Gobierno deLa Rioja-Universidad de La Rioja-CSIC). Servicio deInvestigación y Desarrollo Tecnológico Agroalimentario(CIDA), Ctra. Logroño-Mendavia NA-134 Km. 90,26071 Logroño, La Rioja, Spaine-mail: evapipeal@msn.com

by the cover crops has potential negative effects ongrapevines such as limited vine vigor and yield reduc-tion (Tesic et al. 2007; Ripoche et al. 2011), but theseeffects can vary according to soil fertility, grapevinecultivar, and the cover crop species. However, thesepotentially negative effects can be judged as beneficialin fertile soils where vines have a high vigor. In thosecases, the cover crop can reduce the excessive grapevinevegetative growth that is unfavorable to high-qualitywine production. Moreover, a moderate N status invines reduces the potential for fermentation haze orconditions that can lead to excess thiol formation (Belland Henschke 2005).

In the appellation d’origine contrôlée (AOC) Riojaarea, which hasMediterranean climatic conditions, mostvineyards use tillage for soil-surface management, andthe norm of this appellation limits the red grape yield to6500 kg ha−1. In addition, the soils of La Riojavineyards have a low organic matter content (<1 %)and a loamy texture (Peregrina et al. 2010a), and con-sequently, they are highly susceptible to erosion. Recentstudies indicate that the use of permanent cover crops inAOC Rioja vineyard can increase both organic carbonand stable aggregates in the soil thus improving soilquality (Peregrina et al. 2010b, 2014). Also, cover cropscan decrease the soil N availability (Peregrina et al.2012; Pérez-Álvarez et al. 2013a). Therefore, in theAOC Rioja, cover crops can be an alternative to tillagein order to improve soil quality and also when areduction in vigor and an improvement in the mustquality are needed.

Must quality is influenced by nitrogen compositionwhich affects fermentation kinetics and the productionof ethanol, glycerol, and both aromatic and spoilagecompounds (Albers et al. 1996). Few reports exist onspecific relationships between individual free aminoacids and wine volatiles, but such associations are anarea of active research (Ugliano et al. 2009). The twomain sources of yeast-assimilable nitrogen (YAN) areprimary amino acids and ammonium (Butzke 1998).Consequently, YAN content in grapes can have a sig-nificant impact on the production of many active me-tabolites related to flavor (Hernández-Orte et al. 2005).

Xi et al. (2010) found that in a Cabernet Sauvignonvineyard, cover crops increased the wine aroma com-pounds when compared to tillage. By contrast, Lee andSteenwerth (2011), in a Californian vineyard with sim-ilar Mediterranean climatic conditions, found no signif-icant difference in the YAN composition of Cabernet

Sauvignon grapes under different cover crop manage-ment. However, the different rootstocks used in thatexperiment (110R high vigor, 420A low vigor) playeda significant role in YAN composition.

The reduction of the grapevine N uptake and theconsequent YAN decrease could be positive for winequality when it results in a lower concentration of aminoacids (i.e., histidine) which are precursors of biogenicamines such as histamine. However, if YAN is too low,the fermentation can be negatively affected. When at-mospheric N in the soil is fixed by using a leguminouscover crop, soil N availability and N content in the mustcan increase (Fourie et al. 2007a). The effects of Napplication on grape amino acid composition have beenstudied previously by Conradie (2001) and Linsenmeiret al. (2008). However, little research has been doneconcerning the different effects of gramineous and legu-minous cover crops on soil N availability, N grapevinestatus, and YAN grape composition in cv. Tempranillowhich is the main cultivar in the AOC Riojawinegrowing area. We hypothesized that a nonlegumi-nous cover crop could compete for soil N and decreaseN uptake by the grapevine and consequently affect thegrape YAN content and the production of aromatic andspoilage compounds during fermentation.

The objective of this study was to investigate theinfluence of two different cover crops, namely a gra-mineous [barley (Hordeum vulgare L.)] and a legumi-nous [Persian clover (Trifolium resupinatum L.)] crop,in the midterm (1 to 3 years after cover crop establish-ment) on soil N availability, N grapevine status, andYAN grape content in a highly vigorous cv. Tempranillovineyard in the AOC Rioja.

Materials and methods

Experimental design and site

The experiment began in 2009 in an experimental farmin Nájera, on a terrace of the Najerilla river (468 m ofelevation above sea level), in La Rioja region (northernSpain). Coordinates for the site were lat. 42° 26′ 34″ N;long. 2° 43′ 32″ W. The field slope was about 0.2 %facing west. The soil was classified as OxyaquicXerorthent (according to USDA Soil Taxonomy). Ad-ditional details for soil characteristics are shown inTable 1. The climate in the area is semiarid accordingto the UNESCO aridity index.

Plant Soil

Weather conditions were recorded by a RiojanAgroclimatic Service meteorological station(www.larioja.org/siar) installed near the experimentalvineyard (lat. 42° 27′ 42.75′ N; long. 2° 42′ 45.66′ W;altitude 465 m above sea level). Annual precipitation,average annual temperature, and annual potentialevapotranspiration (FAO-Penman Monteith) from years2009, 2010, and 2011 are represented in the Fig. 1.

The vineyard was planted in 1999 with Vitisvinifera L., cv. Tempranillo, grafted on 110-Richter rootstock. Planting density was 2850 plantsper hectare with vine and row spacing of 1.3 and2.7 m, respectively, rows had a west-east orienta-tion. Vines were trained to a vertical shoot posi-tion trellis system on a double Cordon de Royatand spur pruned to twelve buds per vine. Inspring, all water shoots were pulled off.

The experimental design was a randomized completeblock with three treatments and three replications pertreatment. Each replicate (plot) had four adjacent rowswith 20 vines per row. In each replicate, the two outerrows were considered buffer zones and excluded insampling to avoid edge effects. Three types of soil covermanagement between rows were studied: CT, conven-tional tillage; B, cover crop of barley (Hordeum vulgareL.) cv. Naturel; and CL, cover crop of Persian clover(Trifolium resupinatumL.). Both cover crops were sownin the alley area with a rotary seed drill in winter(February) 2009 and resown in February 2011. Vegeta-tion of clover cover crop was mowed in spring 2010because of the high cover crop production. The biomasswas left on the alley soil surface. The two species sowedwere self-seeding. In order to eliminate weeds, soiltillage alleys were disked (0 to 15 cm depth) every 4to 6 weeks during the growing season (February–Au-gust) in the CT treatment.

Table 1 Soil characteristics of the Ap horizon

Soil characteristics of experimental site

Soil classificationa Oxyaquic Xerorthent

Ap horizon depth (cm) 15

pH (H2O, 1:5) 8.5

ECb 1:5 (dS m−1) 0.11

Organic matter (%) 1.24

Carbonates (%) 4.2

Color munsell (dry) 10 years 5/4

Particle size distribution (%) USDAc

Sand (2000–50 μm) 38.3

Silt (50–2 μm) 43.2

Clay (<2 μm) 18.5

a Soil taxonomy from USDAbEC electrical conductivityc USDA United States Department of Agriculture

Fig. 1 Annual precipitation,average annual temperature, andannual potentialevapotranspiration (FAO-PenmanMonteith) from years 2009, 2010,and 2011

Plant Soil

In all the treatments, a 0.8-m-wide herbicide strip waskept beneath the vines so the cover crop had awidth of 1.9 m.

The experimental vineyard had not been N fertilizedsince 2000 (9 years prior to the experiment starting), sothe N dynamics for cover crops and conventional tillagecould be evaluated. In the three treatments, after prun-ing, canes were shredded and left on the soil surfacebetween rows. The rest of the vineyard managementpractices were similar in all treatments. Grapevine can-opy management and control of vine pests and diseasesby chemical spraying were carried out by machines, andthe crop was manually harvested.

Soil total nitrogen, NH4+-N, and NO3

−-N analysisthroughout the growing season

In each plot, three subsamples were collected randomlyby an auger in the interrow, at 0 to 15 and 15 to 45 cmdepth and bulked to give a composite sample. Fivesamplings were carried out at each growing season; atbudbreak (end-April), bloom (mid-June), fruit set(mid-July), veraison (mid-August), and postharvest(early-November).

The soil samples were air dried and then ground andsieved to 2 mm. The percentage of coarse fragments(>2 mm) was determined in each sample. Soil totalnitrogen (organic N+inorganic N) was determined, insoil samples taken at bloom, by means of a dry com-bustionmethod (Matejovic 1997), using a TruSpec CNSElemental Analyzer (LECO Inc.; St. Joseph, MI, USA).Soil NH4

+-N and soil NO3−-N were extracted from the

soil samples with 2 M KCl and determined by colorim-etry at 660 and 550 nm, respectively, using a SEALAutoAnalyzer 3HR (Seal Analytical; Hamburg, Germa-ny), an elemental autoanalyzer of segmented flow. Inorder to express N content as NO3

−, NH4+, and total N in

kilograms per hectare, soil bulk density determined bythe core method, as well as the soil percentage of coarsefragments (>2 mm) at both 0 to 15 and 15 to 45 cm soildepths were employed.

Determination of leaf nitrogen content

A total of 20 healthy leaves per plot (only one leaf pervine, distributed randomly) were sampled from nodesopposite to the first bunch at bloom (mid-June). Atveraison (mid-August), 20 healthy leaves were alsosampled from nodes opposite to the second bunch.

In each growing season, blades and petioles wereseparated, washed with tap water, and rinsed with dis-tilled water. The plant material sample was dried at 70 °C in a forced-air oven (Selecta Dry-Big; J.P. Selecta,Barcelona, Spain) for 72 h and then ground and sievedto 0.5 mm in an ultracentrifuge mill (ZM1; Retsch,Haan, Germany). Nitrogen concentration in leaf bladesand petioles was determined with an elemental analyzerTruSpec CN (Leco Inc.; St. Joseph, MI, USA). Concen-trations were expressed on a dry weight basis (García-Escudero et al. 2013).

Yield, grape sampling, and grape must

At harvest in 2009, 2010, and 2011, 20 randomly cho-sen vines in each plot were harvested just prior to theAOC Rioja commercial harvest. The grape yield pro-duced per plot and the number of clusters per grapevinewere recorded. At the same time, random samples ofapproximately 500 berries were collected from 20grapevines distributed randomly throughout each plot.In the laboratory, berries were counted, weighed, andcrushed using a Masticator (IUL Instruments; GmbH,Königswinter, Germany) to obtain must which was keptfrozen (−20 °C) until analyses were carried out.

HPLC analysis of free amino acids and YANdetermination

Analysis of amino acids in must was performed accord-ing to the method described by Garde-Cerdán et al.(2014). Briefly, free amino acids (aspartic acid BASP,^glutamic acid BGLU,^ asparagine BASN,^ serineBSER,^ histidine BHIS,^ glycine BGLY,^ threonineBTHR,^ citrulline BCIT,^ arginine BARG,^ alanineBALA,^ tyrosine BTYR,^ valine BVAL,^ methionineBMET,^ tryptophan BTRP,^ phenylalanine BPHE,^ iso-leucine BILE,^ ornitine BORN,^ leucine BLEU,^ lysineBLYS,^ and proline BPRO^) were analyzed by reversephase using a Hewlett Packard Series 1100 LiquidChromatograph (Agilent Technologies; Waldbronn,Germany) equipped with an automatic liquid sampler(ALS), an Agilent 1100 fluorescence detector (FLD),and an ultraviolet diode array detector (DAD). An ali-quot (5 mL) of sample (previously centrifuged,4000 rpm, 10 min) was mixed with 100 μL of norvaline(internal standard to quantify all amino acids exceptPRO) and 100 μL of sarcosine (internal standard toquantify PRO). The mixture was filtered through a

Plant Soil

0.45-μmOlimPeak pore filter (Teknokroma; Barcelona,Spain) and sent to an automatic precolumn derivatiza-tion with o-phthaldialdehyde (OPA Reagent, AgilentTechnologies; Palo Alto, CA, USA) for primary aminoacids determination and with 9-fluorenylmethylchloroformate (FMOC Reagent, Agilent TechnologiesInc.; Palo Alto, CA, USA) for determination of second-ary amino acids. The injected amount from the derivatedsample was 10μL, and temperature was kept constant at40 °C. All separations were performed on a HypersilODS column (250×4.0 mm, I.D. 5 μm) (AgilentTechnologies Inc.; Palo Alto, CA, USA). Solventsand gradient conditions for amino acids analysisare described below.

Two eluents were used as mobile phase: eluent A,75 mM sodium acetate, 0.018 % triethylamine(pH 6.9)+0.3 % tetrahydrofuran; eluent B, water, meth-anol, and acetonitrile (10:45:45, v/v/v). All reagentswere first filtered through Millipore filters (0.45 μm).The gradient profile employed is described in Table 2.Detection was performed by FLD (λ excitation=340 nm, λ emission=450 nm for primary amino acids,and λ exCitation=266 nm, λ emission=305 nm forsecondary amino acids) and DAD (λ=338 nm for pri-mary amino acids and λ=262 nm for secondary aminoacids). Identification of compounds was carried out bycomparing their retention times with those of pure ref-erence standards. The pure reference compounds andinternal standards were from Sigma (Sigma-Aldrich; St.Louis, MO, USA). Water was obtained from a Milli-Qpurification system (Millipore Corporation; Billerica,MA, USA). Quantification of amino acids was per-formed employing norvaline and sarcosine as internalstandards, as mentioned above.

Unit for free amino acid was milligrams N per kilo-gram of whole berries (which will be referred to as mgNkg−1 for conciseness). Total free amino acids (FAN)were determined by summing the individual free aminoacid values.

Yeast-assimilable nitrogen values (mg N kg−1) weredetermined according to the method described by Aerny(1996). Ammonia concentration was calculated by sub-traction from the YAN content, the individual primaryfree amino acids, as recommended by Lee and Schreiner(2010) that can be easily utilized by yeast (all individualfree amino acids excluding HIS and PRO).

Statistical analysis

Treatment effects on measured variables were testedusing ANOVA (univariate linear model), and differ-ences between treatment means were determined usingthe least significant difference (LSD) multiple range testcalculated at P<0.05. The Pearson correlation coeffi-cient (r) was used to assess the relationship between Nconcentration in leaf tissues (blade and petiole) andammonia and free amino acids. Statistical procedureswere carried out with the software program StatgraphicsPlus for Windows (1998) (StatPoint Technologies Inc.;Warrenton, VA, USA).

Results

Cover crops effect on total N, extractable NH4+-N,

NO3−-N availability, leaf N content, and on yield com-

ponents throughout the 3 years monitored.At bloom in 2011, the CL cover crop showed higher

total soil N with respect to the CT treatment. Moreover,the CL cover crop had greater NH4

+-N with respect tothe CT and B treatments in the 0- to 15-cm soil depth in2010 and 2011 seasons (Fig. 2). There were no differ-ences between CT and B treatments for total soil N andNH4

+-N at either depth nor year.From the first soil sampling taken (budbreak, April

2009) to the last (postharvest, November 2011), soilNO3

−-N content in the B treatment was consistentlylower with respect to CT at both 0- to 15- and 15- to45-cm soil depths (Fig. 3a, b), although for the deepersoil layer, differences between treatments were smaller.A few exceptions where differences between treatmentswere not significant occurred for the 0- to 15-cm depthat budbreak and bloom in 2010, and for the 15- to 45-cm

Table 2 Gradient profile of eluent B (water, methanol, and ace-tonitrile in relation 10:45:45, v/v/v), employed in the determinationof amino acids with HPLC

Time (min) Eluent B (%) Flow rate (mL/min)

0 to 15 0 to 47.5 1.63

15 to 15.01 47.5 1.63 to 0.80

15.01 to 25 47.5 to 60 0.80

25 to 25.01 60 0.80 to 1.63

25.01 to 26.01 60 to 100 1.63

26.01 to 26.51 100 1.63 to 2.50

26.51 to 34.01 100 2.50

34.01 to 36.01 100 to 0 1.63

Plant Soil

soil depth at budbreak, bloom and setting in 2009 sea-son, and budbreak in 2010 (Fig. 3a, b).

For both sampling depths, the soil NO3−-N dy-

namic under CL treatment showed a wider rangein values than under B. In 2009, the CL treatmentsat 0- to 15- and 15- to 45-cm soil depths(Fig. 3a, b) had lower soil NO3

−-N contents com-pared to those from the CT treatment. Although at15- to 45-cm soil depth, the difference was onlysignificant from veraison (Fig. 3b).

In contrast, the CL soil NO3−-N content at 0- to 15-

cm soil depth was higher than both CTand B treatmentsat setting and veraison in 2010 and at budbreak, setting,veraison, and postharvest in 2011 (Fig. 3a). At 15- to 45-cm soil depth, also the CL treatment had higher soilNO3

−-N content than both CT and B treatments atsetting, veraison, and postharvest in 2010 and in 2011and also at budbreak in 2011 (Fig. 3b).

Regarding the effect of cover crops on leaf N content(Fig. 4) in 2009 at veraison stage, B cover crop showed areduction in N content in both blade and petiole tissueswith respect to CT treatment. In 2010, at veraison,petiole N content was lower in B than in CT leaf tissuessamples. At bloom, in 2011 season, both blade andpetiole N content were lower in B than in CT and CLtreatments.

Finally, the three yield components measured (grapeyield, berry weight, and cluster per grapevine) did notshow significant differences between treatments in thethree seasons of the experiment (Table 3).

Effect of cover crops on nitrogen-containing com-pounds: individual free amino acids, FAN, ammonia,and YAN.

In the first harvest after the cover crop was sown(2009), grapes of both B and CL cover crop treatmentsshowed a trend of lower content of ammonia, free amino

Fig. 2 Total nitrogen and NH4+-N (kg ha−1) content in 0- to 15-and 15- to 45-cm soil depths from the three treatments: (CT)conventional tillage, (B) cover crop of barley, and (CL) cover crop

of clover at grapevine bloom in 2009, 2010, and 2011 seasons.Vertical bars represent standard error. Different letters indicatesignificant differences between treatments with LSD test (P<0.05)

Plant Soil

acids, and YAN with respect to CT. However, in the2009 season, differences between treatments weresignificant only in the case of GLY for B withrespect to CT (Table 4).

In 2010, the content of ASP, GLU, HIS, CIT, TYR,VAL, MET, TRP, PHE, LEU, and PRO was sig-nificantly lower in grapes from B and CL when

compared to that in grapes from CT. In addition,CL grapes were also lower in SER, GLY, THR,and ILE than CT grapes, and B grapes had alower ASN concentration than CT grapes.

In the third harvest (2011), CT and CL grape extractswere higher in FAN, ASP, SER, THR, TYR,MET, TRP,LYS, PRO, and YAN than those from the B cover crop.

Fig. 3 Evolution of the soil NO3−-N availability (kg ha−1)

throughout the 3 years monitored (2009, 2010, and 2011) frombudbreak (BB), bloom (BL), set (S), and veraison (V) to posthar-vest (P), in 0- to 15- (a) and 15- to 45-cm (b) soil depth in the three

treatments: (CT) conventional tillage, (B) cover crop of barley, and(CL) cover crop of clover. Vertical bars represent standard error.Bars indicate significant differences between treatments with LSDtest (P<0.05)

Plant Soil

Only GLU and GLY concentrations were higher in CTwith respect to B but not statistically different fromthose in CL. Ammonia, ASN, HIS, CIT, ARG, VAL,PHE, and ORN concentrations were higher in CL thanin B (Table 4).

Relationship between leaf N contentand nitrogen-containing compounds in grape extracts

Blade N content at bloom was positively correlated withNH4

+, FAN, GLU, ASN, HIS, GLY, ARG, TYR, ORN,LYS, PRO, and YAN (Table 5). In the case of ASP, itscorrelation with the N blade content was negative. Pet-iole N content was positively correlated with FAN, ASP,

HIS, CIT, ARG, ALA, TYR, MET, TRP, LEU, andYAN. In addition, the relationships between leaf Ncontent at bloom and YAN and free amino acids in themust at harvest were better than the relationships be-tween leaf N content at veraison and YAN and freeamino acids at harvest (data not shown).

Discussion

In our study, neither soil total N nor soil NH4+-N content

in B cover crop differed with respect to CT treatmentand, therefore, cannot explain the reduction of leaf Ncontent and grape amino acids content observed in the B

Fig. 4 Nitrogen content (%, dry matter) in blade and petioletissues from the three treatments: (CT) conventional tillage, (B)cover crop of barley, and (CL) cover crop of clover at grapevine

bloom and veraison in 2009, 2010, and 2011 seasons. Differentletters indicate significant differences between treatments withLSD test (P<0.05)

Plant Soil

treatment with respect to CT. However, in 2011 season,at 0- to 15-cm soil depth, soil with the CL cover croppresented both soil total N and soil NH4

+-N content,higher than soils with CT and B treatments. The greatertotal N content in the soil surface in this third year withCL cover crop was related to the capacity of leguminousspecies to fix N. The higher soil NH4

+-N content withCL cover crop in 2011 season would indicate that ahigher rate of organic N mineralization existed, and thisNH4

+-Nmineralized can be easily transformed to NO3−-

N in the soil thus explaining the higher NO3−-N content

shown under the CL treatment.Moreover, the results of our study show that from the

first season with the treatments, the grapevines grownwith the B cover crop treatment had a lower soil NO3

−-N availability than those grown under a CT soil man-agement. This reduction of soil NO3

−-N availabilitywith the B treatment could be due to the addition ofvarious factors such as N uptake by the cover crop(Pérez-Álvarez et al. 2013a), a lower N mineralizationrate due to the reduction in soil moisture caused by thecover crop (Celette et al. 2009), or a higher NO3

−-Nrecycling by the soil microorganisms under covercrop (Steenwerth and Belina 2008). In agreementwith our results, several authors have reported adecrease in the soil NO3

−-N availability undercover crops with respect to tillage in vineyardswith Mediterranean climatic conditions in differentlocations such as southern France (Celette et al.2009), California (Steenwerth and Belina 2008),and Spain (Peregrina et al. 2012). In addition,Ripoche et al. (2011) reported a reduction of

inorganic soil N at setting with a cover crop in aMediterranean vineyard in France.

The growth and N supply of leguminous cover cropsdepend on the species, the length of growing season,and the weather and soil conditions (Shennan 1992).Also, the amount of mineralizable N supplied by theleguminous cover crop depends on the growth stage atwhich the cover crop is incorporated into the soil (Kuoet al. 1996). Thus, under our conditions, the Persianclover cover crop can increase the soil NO3

−-N avail-ability from the second season after cover crop wasestablished. Fourie et al. (2007a) reported, in SouthAfrican vineyards, a higher inorganic N content underdifferent leguminous cover crops than in conventionaltillage at soil surface (0 to 15 cm).

With respect to the N nutritional status of the grape-vine, leaf N content decreased with the B cover crop atleast in one tissue and phenological stage for each of thethree seasons monitored. This decrease of leaf N contentin B treatment with respect to CT treatment can beexplained by the reduction in the soil N availabilityprovoked by the cover crop competition. This resultagrees with the relationship between soil NO3

−-N avail-ability at bloom and leaf N content found in cv.Tempranillo vineyards in the AOCRioja (Pérez-Álvarezet al. 2013b). Moreover, in the third year with B covercrop, N concentration in both blade and petiole tissuesand at both bloom and veraison stages reached deficientvalues according to reference levels for cv. Tempranilloin the AOC Rioja (i.e., low % N level in blade at bloom<2.96, in blade at veraison <2.08, in petiole at bloom<0.76, and in petiole at veraison <0.43) (García-

Table 3 Yield components:grape yield (kg grapevine−1),weight of 100 berries (g), andnumber of cluster per grapevinefrom the different treatments (CTconventional tillage, B cover cropof barley, and CL cover crop ofclover) in 2009, 2010, and 2011seasons

ns indicates non significant dif-ferences between treatments.

Year Treatment Grape yield(kg grapevine−1)

Weight of 100berries (g)

Number of clusterper grapevine

2009 CT 4.88 ns 289.93 ns 13.81 ns

B 5.52 ns 288.67 ns 14.91 ns

CL 5.49 ns 280.90 ns 15.26 ns

L.S.D. P<0.05 1.50 18.09 2.11

2010 CT 5.91 ns 250.33 ns 16.56 ns

B 5.73 ns 254.57 ns 16.21 ns

CL 6.22 ns 245.80 ns 16.77 ns

LSD P<0.05 1.24 13.81 1.93

2011 CT 5.12 ns 292.20 ns 15.57 ns

B 5.07 ns 289.07 ns 16.69 ns

CL 4.20 ns 278.07 ns 14.10 ns

LSD P<0.05 1.10 14.27 2.66

Plant Soil

Tab

le4

Ammonia,freeam

inoacid,and

yeast-assimilableN(m

gkg

−1)ingrapesextractsfrom

thedifferenttreatments(CTconventio

naltillage,Bcovercropofbarley,and

CLcovercropof

clover)in

2009,2010,and2011

seasons

2009

2010

2011

(mgN

kg−1)

CT

BCL

LSD

,P<0.05

CT

BCL

LSD,P

<0.05

CT

BCL

LSD,P

<0.05

Ammonia

26.75nsw

21.37ns

26.ns

18.29

28.48ns

25.48ns

22.37ns

14.23

16.13ab

7.50b

20.79a

9.38

FAN

89.6

ns79.02ns

86.33ns

17.36

106.99

ns76.84ns

83.16ns

11.03

76.95a

58.41b

79.29a

13.84

ASP

0.70ab

0.77a

0.63b

0.11

2.04a

1.65b

1.70b

0.16

1.28a

1.11b

1.32a

0.06

GLU

4.15

ns3.36

ns4.04

ns0.80

3.61a

3.00c

3.22b

0.22

2.72a

2.21b

2.56ab

0.40

ASN

1.16

ns0.87

ns1.06

ns0.52

1.11a

0.69b

0.84ab

0.36

0.43ab

0.31b

0.44a

0.12

SER

3.67

ns3.38

ns3.54

ns0.60

4.37a

3.98ab

3.69b

0.48

2.29a

1.82b

2.36a

0.40

HIS

5.00

ns3.45

ns4.99

ns2.51

6.24a

1.95b

3.59b

2.40

4.12ab

2.24b

4.29a

2.04

GLY

0.88a

0.60b

0.70ab

0.22

0.68a

0.52ab

0.51b

0.17

0.33a

0.22b

0.31ab

0.10

THR

2.86

ns2.79

ns2.80

ns0.74

3.49a

3.06ab

2.89b

0.52

1.96a

1.43b

2.04a

0.40

CIT

1.06

ns0.87

ns0.87

ns0.61

1.63a

0.84b

0.93b

0.42

0.92ab

0.61b

0.98a

0.33

ARG

26.41ns

22.78ns

25.26ns

4.59

25.97ns

21.92ns

20.92ns

5.58

26.65ab

19.90b

28.28a

8.06

ALA

5.11

ns4.67

ns4.89

ns0.71

5.26

ns4.99

ns4.67

ns0.69

6.64

ns5.60

ns6.91

ns1.51

TYR

0.41

ns0.39

ns0.40

ns0.15

0.49a

0.33b

0.33b

0.09

0.42a

0.28b

0.46a

0.13

VAL

1.65

ns1.87

ns1.72

ns0.38

3.26a

2.38b

2.21b

0.56

1.03ab

0.87b

1.16a

0.27

MET

0.33

ns0.30

ns0.34

ns0.11

0.60a

0.42b

0.36b

0.12

0.31a

0.18b

0.31a

0.12

TRP

2.21

ns2.19

ns2.21

ns0.42

4.18a

3.57b

3.57b

0.32

2.49a

1.92b

2.65a

0.46

PHE

0.43

ns0.44

ns0.44

ns0.13

0.92a

0.67b

0.60b

0.17

0.33ab

0.28b

0.37a

0.07

ILE

0.42

ns0.44

ns0.41

ns0.08

0.92a

0.77ab

0.66b

0.23

0.41

ns0.38

ns0.49

ns0.15

ORN

0.19

ns0.15

ns0.18

ns0.18

0.17

ns0.10

ns1.00

ns0.13

0.18ab

0.14b

0.23a

0.07

LEU

0.71

ns0.66

ns0.69

ns0.19

1.41a

0.99b

0.87b

0.32

0.83

ns0.61

ns0.87

ns0.30

LYS

0.25

ns0.26

ns0.24

ns0.13

0.16

ns0.15

ns0.12

ns0.08

0.26a

0.18b

0.27a

0.06

PRO

32.09ns

28.76ns

30.78ns

7.04

40.47a

24.86c

31.43b

4.94

23.36a

18.14b

23.01a

3.04

YAN

84.34ns

71.71ns

82.04ns

25.86

95.00ns

78.49ns

74.09ns

23.51

69.73a

47.77b

77.07a

10.22

nsindicatesnonsignificantd

ifferences

betweentreatm

ents.

FANfree

aminoacidsnitrogen,A

SPasparticacid,G

LUglutam

icacid,A

SNasparagine,SERserine,H

IShistidine,GLY

glycine,THRthreonine,CIT

citrullin

e,ARGarginine,A

LAalanine,

TYR

tyrosine,V

ALvalin

e,METmethionine,TRPtryptophan,P

HEphenylalanine,ILEisoleucine,O

RNornithine,LEUleucine,LY

Slysine,P

ROproline,YA

Nyeast-assimilablenitrogen

wIn

thesamerow,different

lettersam

ongtreatm

entsexpresssignificantd

ifferences

atP<0.05

usingtheLSD

test

Plant Soil

Escudero et al. 2013). However, this effect did not occurfor CT and CL treatments. Therefore, we consider that aN-deficient status existed in grapevines from B treat-ment at 2011. These results could be due to the fact thatcultural practices did not affect to the grapevine reservesat short term as showed Holzapfel and Smith (2012).Under B cover crop, the grapevine could employ the Nstored as reserves in its tissues (trunk and roots) in thefirst year, maintaining its N nutritional status; but after3 years with a lower soil N availability, the N reserves inthe grapevine could not compensate for this deficit, andthus, differences were found in the leaf N content withrespect to the CT. Tesic et al. (2007) also found lowerpetiole N content at bloom under cover cropwith respectto bare soil in cv. Chardonnay, and Sweet and Schreiner(2010) reported a lower N concentration in blade atbloom under resident vegetation with respect to tillageand a cover crop of a clover mixture in cv. Pinot noir.

Results of the grapevines under CL cover cropshowed a leaf N content similar to those under CTtreatment. The lower soil NO3

−-N availability showedduring the first season by the CL cover crop with respectto CT treatment was compensated by the ability of coverto fix N. The increase of soil NO3

−-N availability withrespect to CT started in the second season with the CLtreatment, provoking that leaf N content in CL reachedvalues similar to those of the CT treatment.

In contrast to our results, Fourie et al. (2007b) foundthat petiole NO3

−-N content at bloom in Chardonnayand Sauvignon blanc cultivars under a broad leaf le-gume cover crop increased with respect to a tilled soil.This different result could be due to the fact that in theexperiment carried out by Fourie et al. (2007b),the vineyard was irrigated while in our experiment,no irrigation was applied to the plots. In ourconditions, water competition of cover crops couldresult in the fact that soil moisture at soil surfaceunder CL cover crop was lower than that underthe CT treatment, as occurred in a vineyard withno irrigation under Mediterranean climatic condi-tions where cover crops reduce the soil moisture inthe surface layer of soil (Celette et al. 2008). Sowhen soil moisture is too low, soil N may not beavailable to the grapevine (González-Dugo et al.2010) and this effect could explain the differencesbetween our results with respect to those fromFourie et al. (2007b).

Regarding the yield components, both cover cropshad no effect on any yield components, indicating thatT

able5

Pearson

correlationcoefficientsandits

Pvalues

betweenleafN(%

N)tissues(blade

andpetio

leatbloom)and

nitrogen-containing(m

gNkg

−1)com

pounds

(NH4+,FAN,Y

AN,and

aminoacids)in

grapeextractsthroughout

the3yearsmonito

redin

thetreatm

ents(CTconventio

naltillage,Bcovercrop

ofbarley,and

CLcovercrop

ofclover)(n=27)

Bloom

NH4+

FAN

ASP

GLU

ASN

SER

HIS

GLY

THR

CIT

ARG

Blade

(%N)

0.4092*

0.4884**

−0.4601*

0.5272**

0.5653**

0.1927

0.5898**

0.5511**

0.2748

0.3726

0.5965**

Petio

le(%

N)

0.3587

0.4599*

0.4058*

−0.0201

0.1131

0.1308

0.4930*

0.0315

0.2086

0.5328**

0.6139**

ALA

TYR

VAL

MET

TRP

PHE

ILE

ORN

LEU

LYS

PRO

YAN

Blade

(%N)

0.1175

0.5710**

−0.0696

0.0972

−0.2294

−0.0982

−0.3661

0.5230**

−0.1421

0.6112**

0.4054*

0.5840**

Petio

le(%

N)

0.5515**

0.5987**

0.1323

0.4689*

0.4068*

0.3126

0.2093

0.2793

0.4508*

0.1979

0.2211

0.5510**

*Significant

correlationatP<0.05;*

*significant

correlationatP<0.01

FANfree

aminoacidsnitrogen,A

SPasparticacid,G

LUglutam

icacid,A

SNasparagine,SERserine,H

IShistidine,GLY

glycine,THRthreonine,CIT

citrullin

e,ARGarginine,A

LAalanine,

TYR

tyrosine,V

ALvalin

e,METmethionine,TRPtryptophan,P

HEphenylalanine,ILEisoleucine,O

RNornithine,LEUleucine,LY

Slysine,P

ROproline,YA

Nyeast-assimilablenitrogen

Plant Soil

differences found in the composition of N-containingcompounds in the grapes, would not be attributable toprocesses of concentration by reducing grape produc-tion or berry weight. In agreement with our results,Ingels et al. (2005) in California and Lopes et al.(2008) in Portugal found that the use of cover crop assoil management did not reduce grapevine yield.

In addition, under our experimental conditions, FANand YAN decreased under B cover crop in the thirdseason, and positive relationships between FAN andYAN with the leaf N content in grapevine at bloomwere found. In agreement with our results, Gaudillereet al. (2003) found correlations between blade N contentand N in must in vineyards under Atlantic andMediterranean climates in France, and Neilsen et al.(2010) described correlations between petiole-N atveraison and yeast-assimilable N in cv. Merlot underdifferent rate, timing, and form of N fertilization. Also,N concentration in must increased under a leguminouscover crop and decreased under a grain cover crop bothwith respect to conventional tillage in cultivars Char-donnay and Sauvignon blanc (Fourie et al. 2007b).Besides this, Lee and Schreiner (2010) found that incv. Pinot noir, lower N supplies reduced the YAN con-tent in grapes. Low YAN content in must could result insluggish fermentation and could produce residual sugar(Spayd et al. 1995). Nitrogen concentrations from 70 to270mgNL−1 YAN are reported to be adequate to insuregood fermentation (Bell and Henschke 2005). There-fore, in our experimental vineyard where CT had a YANconcentration near to the aforementioned minimum, thedecrease of YAN concentration in the third season underthe B cover crop could be undesirable for an adequatefermentation. From these results, studies could bedeveloped for a practical application in thevineyards, such as applying N fertilizer afterbloom with fertilizer rates calculated in functionto the leaf N content at this phenological stage,with the objective to insure an adequate level ofN-containing compounds in the must at harvest.

With respect to the grape amino acids profile in thedifferent treatments, we found a total of 20 free aminoacids with PRO and ARG being dominant. In our re-sults, the amino acid profile was modified by soil man-agement with the cover crops because 15 amino acidshad positive correlations with leaf N (blade or petiole)content at bloom. However, the N content in both bladeand petiole tissues at bloom did not correlate with thesame group of amino acids (i.e., GLU, ASN, GLY,

ORN, LYS, and PRO only was correlated with blade,and CIT, ALA, MET, TRP, and LEU only was correlat-ed with petiole). Therefore, blade or petiole tissue couldbe used depending on the specific amino acids to studyin grapes. Recently, Romero et al. (2013) reported thatblade, at both bloom and veraison stages, was the mostsuitable tissue to N nutrition diagnosis in the cv.Tempranillo in AOC Rioja. However, according to ourresults, the petiole should also be considered for thediagnosis of N-containing compounds (such as aminoacids) in the grapes.

Positive relations between the N supply and the freeamino acids concentration (ALA, HIS, GLY and GLU)were reported in cv. Riesling by Linsenmeir et al.(2008). Lee and Schreiner (2010) showed that onlyGLU, GLN, TRP, and HYP were not affected by Nsupply in cv. Pinot noir. The differences in those refer-ences with respect to our results between the free aminoacids that were affected byN availability could be due tothe fact that the amino acid profile and concentration canvary according to grape cultivar, rootstock, site,seasonal conditions, and level of maturity (Belland Henschke 2005).

Finally, the lower soil N availability and consequent-ly, the lower N status in the grapevine under B covercrop provoked lower concentrations of ASP, GLU,SER, GLY, THR, ARG, TYR, MET, TRP, LYS, andPRO in grapes from the B cover crop with respect to CTafter three seasons. This result could have relevant im-plications on wine quality since HIS, TYR, ORN, LYS,PHE, ARG, and TRP are precursors, by microbial de-carboxylation, of the biogenic amines histamine, tyra-mine, putrescine, cadaverine, 2-phenethylamine,agmatine, and tryptamine, respectively (Vincenziniet al. 2009). Therefore, the use of a B cover crop couldreduce the concentration of biogenic amines and im-prove the wine quality.

In contrast, after three seasons, the CL cover crop hadsimilar concentrations of free amino acids in grape withrespect to CT. This result is in agreement with the factthat B treatment decreased both the leaf N content andthe concentration of various free amino acids. In thisway, as the grapevine N status in the CL treatment didnot vary with respect to CT, the amino acid concentra-tions were, consequently, not affected.

Therefore, under our experimental conditions, the CLcover crop did not show any negative implicationslinked to a higher potential production of biogenicamines in the wine.

Plant Soil

Conclusions

Nitrogen availability as total N or extractable NH4+-N

showed a low sensitivity to the different soil manage-ment practices, and therefore, those parameters cannotexplain the changes in the grapevine N nutritional statusnor in the concentration of both YAN and free aminoacids in the grapes.

However, the two different cover crops studied mod-ify the soil N availability measured as NO3

−-N, althoughthey did so differently. The barley cover reduced theavailability of NO3

−-N in the soil with respect to con-ventional tillage from the first year in which it wasestablished; whereas, with respect to conventional till-age, clover cover reduced NO3

−-N when it wasestablished, but increases it afterwards.

These changes in the soil N availability affected thevine nutritional status and also the concentration of bothYAN and free amino acids in the grapes, which candirectly affect the biogenic amines biosynthetic path-way. Nevertheless, these changes show a delay (twoseasons) with respect to the observed change in the soilN availability.

YAN reduction under the barley cover crops cancause defective alcoholic fermentation of musts. How-ever, the lower concentration of amino acids which areprecursors of biogenic amines could improve the poten-tial quality of the wine. Meanwhile, the clover cover didnot have these effects because YAN and FAN concen-trations were similar to the ones found in the conven-tional tillage treatment.

Therefore, our study offers new knowledge showingthe implications of cover crops management. A gramin-eous cover crop such as barley can be suitable to reducethe N grapevine status and both YAN and freeamino acids in the grapes, although this effectwould occur in the midterm (3 years), but not inthe short term (1 year).

Acknowledgments This study was supported by the SpanishMinistry of Science, INIA (Instituto Nacional de Investigación yTecnología Agraria y Alimentaria) through project INIA-RTA2009-00101-00-00 and European Social Fund. F. Peregrina andT. Garde-Cerdán thank the INIA and European Social Fund fortheir postdoctoral grants and E. Pérez-Álvarez thanks the INIA forher predoctoral grant.

We particularly thank Ricardo Leza for lending us the vineyardin which we installed the experimental plots and for helping withthe field labor. Also, we thank Rosa López Martín (ICVV-CIDA,Government of La Rioja) for her help with the analysis method-ology of amino acids in grapes.

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