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South African Journal of Enology and Viticulture

On-line version ISSN 2224-7904
Print version ISSN 0253-939X

S. Afr. J. Enol. Vitic. vol.40 n.2 Stellenbosch  2019 



Nitrogen and Sulphur Foliar Fertilisation



F.A. Bruwer; W. du Toit; A. Buica*

Department of Viticulture and Oenology, P/Bag X1 Matieland, Stellenbosch 7600, South Africa




The effects of fertilisation can reverberate from grapes through to wines. In wine, non-volatile compounds mainly influence the taste and flavour of wines, while volatile compounds play an important role in the aromatic expression. This review includes information on the presence of non-volatile and volatile compounds reportedly affected by nitrogen and sulphure foliar fertilisation, (bio)synthesis, and evolution throughout winemaking, their chemical properties, and their implications. The second part presents the status of the research on elucidating the influence and contribution of foliar fertilisation practices on the chemical compounds throughout winemaking, from the grape to the wine.

Key words: Foliar fertilisation, nitrogen, sulphur, Chenin blanc, Sauvignon blanc, yeast assimilable nitrogen (YAN), glutathione, thiols




A wine's aroma, taste and flavour can contribute to the wine's overall quality and determine if winemakers and consumers find it appealing (Marais, 1994). The aroma of a wine is the result of various interactions between different chemical compounds found in the wine. These compounds are generated at different stages and through various processes; some compounds originate from the grapes, while others are generated during fermentation or wine ageing. The composition of a grape berry depends on various factors, such as grape variety, environmental conditions, viticultural practices, and terroir. Monoterpenes and methoxypyrazines are derived from the grape, while volatile thiols, esters, higher alcohols and fatty acids are released by yeast from their precursors during alcoholic fermentation (Fischer, 2007).

The nutrient requirements of a grapevine depend on its age, cultivar variety, yield, soil type and properties (Holzapfel & Treeby, 2007). A vine nutrition deficiency occurs often and due to various reasons, and negatively affects the aroma profile of a wine due to sluggish or stuck fermentation (Monteiro & Bisson, 1991). Nutrition deficiencies can be corrected by carrying out fertilisation applications. Foliar nitrogen and sulphur fertilisation applications can positively influence the levels of nitrogen and sulphur compounds in the must, the success rate of alcoholic fermentation, and the resulting wine's composition and aroma (Lacroux et al., 2008; Dufourcq et al., 2009; Lasa et al., 2012; Hannam et al., 2014; Geffroy et al., 2016a; Dienes-Nagy et al., 2017; Gutiérrez-Gamboa et al., 2017b; Helwi et al., 2017; Kelly et al., 2017). Results obtained through aroma and chemistry research in foliar fertilisation studies can contribute to the knowledge base of aromas and chemical compounds of specific cultivar varieties.

The first part of this literature review will focus on various wine compounds and classes of compounds present in grapes and wines that were shown to be affected by foliar fertilisation. The second part will present the status of the research on elucidating the influence and contribution of different fertilisation practices in relation to the chemical compounds of the grapes, juice and wine, but also on the resulting wine's aromatic expression.



Standard oenological parameters (pH, total acidity, Balling and percentage ethanol)

In the past, the determination of harvest date did not include parameters such as pH and TA, and nowadays relying solely on sugar level is not considered a very accurate index to follow (Du Plessis & Van Rooyen, 1982). Optimal grape ripeness can be determined by including the levels of °B, pH, TA and colour of the grape skin and seeds (Deloire, 2012). Van Schalkwyk and Archer (2000) reported various optimal grape ripeness ranges for different classes of South African wines. The sugar content of white wine grapes at harvest ranges from 19.5 to 23°B, while grapes for sparkling wines are harvested at lower levels (18 to 20°B). Red wines nowadays fall within the same range as white wine, or can be slightly higher, and sweet and dessert wine grapes are harvested at higher sugar levels (22 to 26°B) (Van Schalkwyk & Archer, 2000). The pH ranges for white, red and sweet wine grapes are similar (3.2 to 3.4), while sparkling wines have lower levels (2.8 to 3.2), and dessert wines have higher levels (3.3 to 3.7) (Van Schalkwyk & Archer, 2000). The TA levels of red (6.5 to 7.5 g/L), sweet (6.5 to 8 g/L), and dessert wine grapes (6.5 to 8 g/L) are lower compared to those of sparkling wines, at 7 to 9 g/L, and white wines, which range from 7 to 8 g/L (Van Schalkwyk & Archer, 2000).

During winemaking, methods such as skin contact, maceration and pressing can influence the pH and TA levels in the must and juice. Free-run juice results in higher sugar and lower acid levels compared to pressed juice (Amerine et al., 1982). The method of adding tartaric acid to wines to lower their pH levels is effective and should be performed before the start of alcoholic fermentation (Pambianchi, 2001). During alcoholic fermentation, the yeast converts the sugar into alcohol and carbon dioxide (CO2). If the acidity is below 3 g/L, the alcoholic fermentation is somewhat reduced; on the other hand, low pH levels help to inhibit the growth of undesirable bacteria (Amerine et al., 1982). During alcoholic fermentation, the alcohol content increases and the solubility of the acids decreases; therefore TA levels decrease and cause the pH to increase (Pambianchi, 2001; Robinson & Harding, 2014). Cold and protein stabilisation of white wine is done generally at -4°C for a minimum of two weeks. During this procedure, tartaric acid precipitates as potassium bitartrate salt, and the TA of the resulting wine decreases and the pH increases further (Pambianchi, 2001; Robinson & Harding, 2014).

The initial sugar level of the harvested grapes and alcohol levels of the resulting wine are correlated. In South Africa, the residual sugar levels differ for different classes of wines: sparkling wines are from < 3 to > 50 g/L, dry still wines are < 5 g/L), semi-dry wines are > 5 to < 12 g/L), semi-sweet wines vary from > 12 to < 30 g/L), late harvest wines are > 20 g/L, natural sweet wine is > 20 g/L), and noble late harvest wines are > 50 g/L (WOSA, 2017). During wine maturation in barrels, the wine diffuses through the small oak pores, and alcohol diffuses slower than water. Thus, in a dry cellar, the ethanol strength can increase, while in a humid cellar, the ethanol strength will decrease. It is therefore important to top up the barrels regularly during maturation (Robinson & Harding, 2014).

Non-volatile compounds

Various non-volatile compounds are constituents of grapes, juices and wines. These compounds are not aromatic, but some can be considered precursors to aromatic compounds, while others can influence the perception of aroma compounds through interactions. The compounds reviewed in this section include normal oenological parameters, ammonia and amino acids, and glutathione (GSH). Standard oenological parameters, including pH, total acidity (TA), sugar content (Balling) and ethanol content, are commonly measured during the winemaking process. Knowing the nitrogen levels of the must is crucial for successful alcoholic fermentation, and therefore the yeast assimilable nitrogen (YAN) levels are measured. GSH includes the reduced and oxidised forms and can be considered an indicator of the oxidation status.

Yeast assimilable nitrogen

Grapes contain various nitrogen compounds that are grouped into two forms: mineral (NH4+, NO3- and NO2-) and organic (free amino acids, nucleic acids, proteins, ethyl carbamate and urea) (Kuhn et al., 2014). Grape juice and must contain various nutrients and it is important to know which nitrogen compounds are abundant and are required by the yeast for metabolism (Henschke & Jiranek, 1993). The major sources of nitrogen utilised by yeast are known as YAN, and the constituents thereof are free amino nitrogen (FAN) and ammonia nitrogen (NH3). Bell (1994) reported that, during grape ripening, there is a gradual increase in total nitrogen and amino acid nitrogen levels in Cabernet Sauvignon grapes, while ammonium levels decrease. Similar increases and decreases during ripening have been reported by other researchers (Kliewer, 1968; Löhnertz & Schaller, 1992; Hilbert et al., 2003). Henschke and Jiranek (1993) reported that the nitrogen content of grape juice ranges from 60 to 2 400 mg N/L. The yeast assimilable amino acid nitrogen is distributed in different parts in the berry: 10 to 15% in the seed, 19 to 29% in the skin, and 61 to 65% in the pulp (Stines et al., 2000). The assimilable nitrogen content of must provides a good estimation of the vine nitrogen status (Van Leeuwen et al., 2000).

YAN measurements should ideally be performed directly on the must or juice just before alcoholic fermentation to get the most representative results (Bell & Henschke, 2005). Juice samples can underestimate the total berry YAN because the majority of amino acids are contained in the skins of the grape (Stines et al., 2000). Grapes from vineyards with a history of low YAN levels could be analysed for YAN before harvest to get an indication of the levels. In such cases, nitrogen supplementation with di-ammonium phosphate (DAP) at the start of alcoholic fermentation is suggested. Research studies recommend that the minimum YAN level required by the yeast before alcoholic fermentation is between 140 and 150 mg N/L (Henschke & Jiranek, 1993; Spayd et al., 1993; Bell & Henschke, 2005). Lower YAN levels increase the risk of having slow, lagging or stuck alcoholic fermentations, and also the risk of producing hydrogen sulphide (Henschke & Jiranek, 1991). Even though 140 to 150 mg N/L is seen as the critical value for YAN, nitrogen levels practically should be increased to at least 200 mg N/L for a successful fermentation (Leonardelli, 2013). It has been suggested that the nitrogen requirement of yeast differs according to the sugar content of the juice: < 21°B (200 to 250 mg N/L), 21 to 23°B (250 to 300 mg N/L), 23 to 25°B (300 to 350 mg N/L), and > 25°B (350 to 400 mg N/L) (Wilton, 2015).

Ammonia nitrogen

Ammonia nitrogen (NH3, or NH4+ for the ionic form, ammonium) is an important component of YAN. As mentioned above, the ammonia nitrogen concentration declines over time as the grape ripens (Bell, 1994). Various researchers have reported the percentage of ammonia nitrogen of the total YAN content found in berries and juices from different cultivars (Bell, 1994; Spayd et al., 1994; Conradie, 2001; Ribéreau-Gayon et al., 2006; Petrovic et al., 2019). Of the total YAN, ammonia nitrogen levels varied from 32 to 80% in the berries and from 9 to 40% in the juice (Huang & Ough, 1989). Ammonia nitrogen is readily assimilated by the yeast, and is the most preferred nitrogen source during alcoholic fermentation (Monteiro & Bisson, 1991; Henschke & Jiranek, 1993). At the end of alcoholic fermentation, the ammonia nitrogen levels are usually depleted and it is therefore important to know the levels in the grape must before alcoholic fermentation (Ribéreau-Gayon et al., 2006).

The major source of ammonia nitrogen is the berry itself, but additions of DAP to deficient musts can influence the levels thereof and also the total YAN concentration (Henschke & Jiranek, 1991, 1993; Monteiro & Bisson, 1991). Henschke and Jiranek (1993) reported a range of 5 to 325 mg N/L ammonia nitrogen in grapes, while a recent study on South African grape musts has shown that ammonia nitrogen levels ranged from 0 to 165 mg N/L over three seasons (Petrovic et al., 2019).

Free amino nitrogen

FAN includes free or primary amino acids, while secondary amino acids do not fall under this group. All amino acids contain the carboxyl (-COOH) and amino (-NH2 or -NH-) functional groups (Ribéreau-Gayon et al., 2006). From a structural point of view, the difference between primary and secondary amino acids is due to the level of substitution of the N in the amino group; in this case, -NH2 is for primary and -NH- for secondary amino structures (Ribéreau-Gayon et al., 2006).

Amino acids are the most prevalent form of total nitrogen in grape juice and wine. Amino nitrogen distribution in Riesling and Cabernet Sauvignon berries is 10 to 15% in the seeds, 19 to 29% in the skin, and 61 to 65% in the pulp (Stines et al., 2000). The total FAN levels vary in grapes and grape juice depending on the year, and amino acids usually represent 30 to 40% of the total nitrogen in ripe grapes (Ribéreau-Gayon et al., 2006) and 51 to 92% of juice YAN at harvest (Bell, 1994; Spayd et al., 1994; Conradie, 2001).

Several factors can influence the amino acid composition and concentration levels in grapes, including the grape cultivar, rootstock, site, seasonal conditions, and viticultural management (Kliewer, 1968; Etievant et al., 1988). Bell and Henschke (2005) compiled a list of all the amino acids found in whole grapes and/or juice at harvest. During the growth phase of alcoholic fermentation, yeast metabolises grape amino acids, while others are produced by the enzymatic degradation of proteins and some are excreted by live yeasts at the end of fermentation (Lehtonen, 1996). Under anaerobic conditions, amino acids are not metabolised by the yeast during alcoholic fermentation (Duteutre et al., 1971; Ingledew et al., 1987; Long et al., 2012).

Nitrogen compounds, including amino acids, contribute to the formation of compounds like esters, higher alcohols, hydrogen sulphide (H2S), monoterpenes and volatile thiols during the winemaking process (Henschke & Jiranek, 1993; Bell & Henschke, 2005). Various volatile compounds are formed from amino acids during alcoholic fermentation, therefore amino acids can be considered precursors of certain aroma compounds (Henschke & Jiranek, 1993; Bell & Henschke, 2005).

Amino acids can be divided into different groups based on their role or structure: yeast-preferred, branched, sulphur-containing, and other amino acids (Godard et al., 2007; Ljungdahl & Daignan-Fornier, 2012). Yeast-preferred amino acids (aspartic acid (ASP), glutamic acid (GLU), asparagine (ASN), serine (SER), arginine (ARG), alanine (ALA) and glutamine (GLN)) are considered the most important because yeast metabolises them first (Monteiro & Bisson, 1991; Godard et al., 2007). ARG and proline (PRO) are usually the most abundant amino acids in grapes, and ARG is preferred by the yeast as a nitrogen source (Garde-Cerdán & Ancín-Azpilicueta, 2008; Holzapfel et al., 2015). The addition of DAP to juice inhibits the ARG utilisation, and ARG will only be used after ammonia nitrogen has been metabolised by the yeast. Keto acids, such as pyruvic and α-ketoglutaric acid, bind to sulphur dioxide (SO2), react to phenols during winemaking and are formed during the Ehrlich pathway by the amino acids ALA and GLU (Ough et al., 1990).

Branched amino acids (valine (VAL), leucine (LEU), phenylalanine (PHE) and isoleucine (ILE)) are precursors of volatile esters (Antalick et al., 2014). Higher alcohols are formed during the Ehrlich pathway from these amino acids, but a greater proportion are synthesised from sugars (Bell & Henschke, 2005). The relationship between higher alcohols and amino acid assimilation during the fermentation cycle is not clear (Bell & Henschke, 2005). The acids, along with these alcohols, can form esters such as isoamyl acetate and phenylethyl acetate. These amino acids are accumulated in the early stages of alcoholic fermentation and do not support and contribute to the high growth rates during fermentation. The majority of esters are enzymatically synthesised by the yeast from alcohols and medium- and long-chain fatty acids through esterification reactions (Lambrechts & Pretorius, 2000).

Sulphur-containing amino acids are methionine (MET), cysteine (CYS) and cystine (Cys-Cys). They are involved in yeast metabolism under certain conditions and can result in H2S production (Henschke & Jiranek, 1991; Giudici & Kunkee, 1994). When MET becomes depleted in the early stages of alcoholic fermentation, the sulphate reduction sequence (SRS) pathway is activated to reduce sulphate to H2S and to release a surplus thereof alongside mercaptans from the cell (Bell & Henschke, 2005). DAP additions before or during alcoholic fermentation can inhibit the production of H2S (Bell & Henschke, 2005). GSH is formed during alcoholic fermentation from glutamate, GLY and CYS (Castellarin et al., 2012). Research done by Elskens et al. (1991) and Hallinan et al. (1999) suggests that GSH can be degraded to CYS and finally to H2S under nitrogen-deficient conditions. Volatile thiols are found in small amounts in juice must, and their precursors, such as non-volatile, non-glycosylated, odourless S-CYS conjugates, have been identified (Tominaga et al., 1998a, 1998b, 1998c). During alcoholic fermentation, the yeasts degrade the S-CYS thiol precursors to release the volatile thiols (Tominaga et al., 1998a, 1998b, 1998c; Murat et al., 2001a).

All other amino acids (aminobutyric acid (GABA), lysine (LYS), threonine (THR), glycine (GLY), tyrosine (TYR), tryptophan (TRP), histidine (HIS) and ornithine (ORN); hydroproline (HYP) and PRO are secondary amino acids and are not included in the FAN value) are used by the yeast only in the case that other nitrogen sources are depleted first (Duteutre et al., 1971). PRO is among the two dominant amino acids that make up the bulk of the total amino acids (Kliewer, 1968). PRO cannot be assimilated by yeast in the absence of oxygen; therefore, at the end of alcoholic fermentation, the PRO levels have not changed or decreased and high levels are still found in the resulting wine (Ribéreau-Gayon et al., 2006).


GSH was first discovered by Cheynier et al. (1989) in red and white French grapes. It is a sulphur-containing tri-peptide (γ- glutamyl- L- cysteinyl- GLY) and occurs as a natural antioxidant in grapes and must (Anderson, 1998). GSH is formed from the amino acids GLU, GLY and CYS, and contains a nucleophilic -SH centre (Anderson, 1998; Castellarin et al., 2012). GSH is synthesised enzymatically in the grape berry, and the reduced GSH (GSH-R or simply GSH) form is the most abundant thiol-containing compound present at harvest. Suklje et al. (2012) reported that, of the total GSH content in grapes, more than 90% is present in its reduced form.

During crushing, GSH needs to be present in high levels in the must to protect it fully (Singleton et al., 1985). Du Toit et al. (2007) and Kritzinger (2012) report that GSH can be used as a marker of oxidation in winemaking due to its sensitivity to oxidation. GSH reacts with polyphenol o-quinones in juice and wine and consequently limits the effect of oxidation (Cheynier et al., 1989; Makhotkina et al., 2014). Being reactive to quinones, GSH plays an important role in juice by protecting various volatile thiols from oxidation (Chone et al., 2006), and also stopping the formation of unstable aromas in wine (Papadopoulou & Roussis, 2001).

The electrophilic -SH group of GSH-R is reactive, leading to the formation of 2- S- glutathionyl- caftaric acid or grape reaction product (GRP1) (Singleton et al., 1985). The o- quinone of caffeic acid reacts most readily with GSH-R, and phenols can form derivatives of GSH. The GRP is colourless and traps the o-quinone, preventing the must from browning and other reactions from taking place (Kritzinger et al., 2013). The GRP can further be oxidised by laccase and yields an o-quinone, which can brown the polymers and also produce 2,5- di- sulphur- glutathionylcaftaric acid (GRP2) by the addition of another GSH (Singleton et al., 1985; Cheynier et al., 1989). GSH can also react with oxygen and undergo oxidation, resulting in the oxidised GSH form (GSH-O or GSSG).

The GSH concentration increases during grape ripening, and the highest levels occur at the start of véraison (Adams & Liyanage, 1993). Due to enzymatic and redox reactions, GSH levels decrease rapidly during crushing (Adams & Liyanage, 1993). Free-runjuices during pressing are reported to have higher GSH concentrations compared to other higher press fractions (Patel et al., 2010). Levels of GSH-R during alcoholic fermentation usually decrease, and this can be due to the metabolism of the yeast (Du Toit et al., 2007). During wine ageing, GSH (unspecified GSH-R or GSH-O) concentrations decrease (Lavigne et al., 2007; Ugliano et al., 2011). In South Africa, GSH-R concentrations are present in ranges from 1 to 71 mg/L in juice and up to 35 mg/L in wine (Du Toit et al., 2007; Janes et al., 2010; Fracassetti et al., 2011). Reported GHS-O levels found in South African Sauvignon blanc juices ranged from 0.46 to 2.93 mg/L (Du Toit et al., 2007).

Volatile compounds

In addition to the grape-derived aroma compounds, the metabolism of sugar by the yeast during alcoholic fermentation leads to the formation of volatile compounds, such as esters, fatty acids and higher alcohols (Francis & Newton, 2005). These compounds arise as primary metabolites of yeast and sugar and the metabolism of amino acids (Henschke & Jiranek, 1993; Swiegers et al., 2005). The nitrogen status of the must also contributes to the formation of these compounds (Henschke & Jiranek, 1993), but a too-high nitrogen content can reduce the production thereof. The odour thresholds for major volatiles are measured in mg/L, while methoxypyrazines and volatile thiols are much lower and measured in ng/L in juice and wine.

Major volatile compounds


Esters are an important group of volatile compounds and contribute to the pleasant 'fruity' and 'floral' aromas in wines (Swiegers et al., 2005). Esters can be grouped into two groups, namely acetate esters and ethyl esters. The most significant esters are ethyl ethanoate (ethyl acetate), 3-methylbutyl acetate (isoamyl acetate), 2-methylpropyl ethanoate (isobutyl acetate), ethyl hexanoate (ethyl caproate) and 2-phenylethyl acetate (phenethyl acetate) (Thurston et al., 1981). Ethyl esters contribute 'apple' aromas, while acetate esters are associated with 'fruity' aromas (Saerens et al., 2008). Yeast plays a crucial role in the formation of esters. Esters are produced by the metabolism of yeast through lipid and acetyl-CoA metabolism, through the action of alcohol acetyl transferase, an alcohol and a coenzyme A-activated acid condensate (Lambrechts & Pretorius, 2000).

Various factors, such as yeast strain, clarification, and temperature during the winemaking process can influence the levels of esters present in wines. During ageing, ester concentrations can decrease due to hydrolysis or oxidation, and the resulting wines can experience a loss of 'fruity' aromas (Marais, 1978). During bottle maturation of white wine, loss of 'fruitiness' can be linked to the loss of acetate esters, and they tend to diminish more rapidly than ethyl esters (Ramey & Ough, 1980). Ethyl acetate concentrations are much higher compared to those of other esters found in wine.

Fatty acids

Fatty acids contribute to the wine aroma and have an important impact on wine quality (Bell & Henschke, 2005). The most abundant fatty acids in wine include acetic, decanoic, hexanoic, octanoic and decanoic acid. Volatile fatty acid composition ranges from 500 to 1 000 mg/L, and acetic acid, ranging from 0.2 to 2 g/L, accounts for more than 90% of the fatty acids. Generally, red wines have higher concentrations of these acids than white wine (Lambrechts & Pretorius, 2000). Low concentrations can contribute positively to the complexity and aroma of wines (Coetzee, 2011), while unwanted flavours, such as 'cheesy', 'vinegar' and 'rancid', are due to too high concentrations of fatty acids (Lambrechts & Pretorius, 2000). Bacterial spoilage can be linked to elevated levels of acetic acid (Ribéreau-Gayon et al., 2006).

During the early stages of alcoholic fermentation and the biosynthesis of long-chain fatty acids, medium-chain fatty acids like hexanoic, octanoic and decanoic acid are produced as intermediates. Acetic acid is formed as a metabolic intermediate in the synthesis of acetyl-CoA from pyruvic acid, or is formed directly from acetaldehyde by aldehyde dehydrogenases (Bell & Henschke, 2005). Long-chain unsaturated fatty acids, such as oleic and linoleic acid, can enhance alcoholic fermentation, but are not yeast-derived products and originate from the waxy cuticle of grape skins (Lambrechts & Pretorius, 2000). Among the many factors that can influence the fatty acid levels are yeast strain, sugar concentration, inoculation rate, juice clarification, fermentation temperature, nitrogen, oxygen exposure and SO2 additions (Henschke & Jiranek, 1993; Garde-Cerdán et al., 2009; Coetzee, 2011). The nitrogen concentration levels of the must play a crucial role in the volatile acidity in wine.

Contradictory results, related to increases, decreases or stability of certain fatty acids, have been reported during ageing (Roussis et al., 2005; Blake et al., 2009; Lee & Steenwerth, 2011).

Higher alcohols

Higher alcohols, also known as fusel alcohols, are secondary yeast metabolites and can positively or negatively influence the aroma of the wine (Bell & Henschke, 2005). Higher alcohols have more than two carbon atoms in their structure and can be grouped into two categories: aliphatic and aromatic alcohols (Lambrechts & Pretorius, 2000). Aliphatic alcohols include pentan-1-ol (amyl alcohol), 3-methylbutan-1-ol (isoamyl alcohol), 2-methylpropan-1-ol (isobutanol) and propan-1-ol (propanol), while aromatic alcohols include 2-phenylethanol (phenylethyl alcohol) and 4-(2-hydroxyethyl) phenol (tyrosol).

Higher alcohols are formed in two ways during alcoholic fermentation. Firstly, by being synthesised anabolically from intermediates of the sugar metabolism (e.g. glucose), and secondly by being synthesised catabolically from branched amino acids, such as LEU, ILE, THR and VAL (Boulton et al., 1996; Dickinson et al., 2003). Alcohols are formed via the Ehrlich pathway, and the branched amino acids are deaminated and α-keto-acids are decarboxylated and reduced to the corresponding alcohol (Bell et al., 1979).

Must containing high levels of amino acids will produce higher levels of higher alcohols (Swiegers et al., 2005). Concentrations below 300 mg/L add positively to the complexity of wines, whereas more than 400 mg/L can have a detrimental effect and display unpleasant 'fusel' and 'solvent- like' aromas, with the exception of 2-phenyl ethanol ('rose' and 'floral' aromas) (Lambrechts & Pretorius, 2000). Isoamyl alcohol is usually found in wines, with the highest levels ranging from 45 to 490 mg/L at the end of alcoholic fermentation (Lambrechts & Pretorius, 2000). During ageing, alcohols can be oxidised to form aldehyde, causing the concentration levels to decrease (Marais & Pool, 1980).


Methoxypyrazines are nitrogen-containing compounds that are derived from grapes and are situated in the skin and exocarp of grape berries (Marais, 1994). Methoxypyrazines are formed by the catabolism of secondary amino acids, such as VAL, GLY and MET present in the grape (Cheng et al., 1991). Three methoxypyrazine compounds have been identified in Sauvignon blanc, namely 2-isobutyl-3-methoxypyrazine (IBMP), 2-sec-butyl-3-methoxypyrazine (SBMP), and 2-isopropyl-3-methoxypyrazine (IPMP) (Lacey et al., 1991; Marais, 1998).

The accumulation of methoxypyrazines in the berries can be influenced by various factors, such as environmental parameters, clone, canopy management, soil and terroir (Swiegers et al., 2006). During berry ripening, methoxypyrazine levels increase and the highest levels are obtained at véraison, but the levels also start to decrease during véraison (Lacey et al., 1991; Swiegers et al., 2006). Lacey et al. (1988) reported that higher climatic temperatures will lead to lower methoxypyrazine concentrations due to their degradation during ripening and their sensitivity to sunlight, while higher concentrations are obtained in cooler climates. Various research studies link vines with excessive growth to the production of grapes with higher methoxypyrazine concentrations before harvest, irrespective of cluster light exposure. The majority (95.5%) of methoxypyrazines are located in the skin of grapes at harvest time (Roujou de Boubee et al., 2000). The highest levels of IBMP are found in the free-run juice, and increased levels are also found after skin contact (Marais, 1998; Marais et al., 1999; Roujou de Boubee et al., 2000). Methoxypyrazine concentrations can decrease due to clarification processes, while the levels are reported to be stable during alcoholic fermentation (Sala et al., 2004; Kotserides et al., 2008). It can be concluded that viticultural practices can influence the methoxypyrazine concentrations much more than winemaking practices (Roujou de Boubee et al., 2000).

Methoxypyrazine sensory detection thresholds in water are very low: at 2 ng/L for IBMP, 1 ng/L for SBMP and 2 ng/L for IPMP (Lacey et al., 1991; Alberts et al., 2009). Methoxypyrazines are known to contribute to the 'green' aromas present in Sauvignon blanc wines. IBMP contributes to the 'herbaceous' and 'green pepper' aromas, while SBMP contributes to the 'asparagus' and 'green beans' aromas, and IPMP is associated with 'pea' and 'bell pepper' aromas (Ebeler & Thorngate, 2009). Wines with high methoxypyrazine levels can be perceived negatively if the aroma notes are not in balance with other compounds. IBMP is the methoxypyrazine that is found with the highest concentration in Sauvignon blanc musts and wines, while IPMP and SBMP are found at much lower concentrations (Lacey et al., 1991). Van Wyngaard (2013) performed a survey of South African Sauvignon blanc wines and found that the levels of IBMP ranged from 0.4 to 44 ng/L.

Volatile thiols

Volatile thiols are sulphur-containing compounds that are present in wine at very low concentrations and the aromas they produce are powerful. Volatile thiols are generally known to contribute to the positive tropical aroma characteristics of Sauvignon blanc wines, such as 'citrus', 'gooseberry', 'grapefruit', and 'passion fruit' (Tominaga et al., 1998c, 2000). These compounds are sulphur-containing substances with additional functional groups, such as alcohol, ester or ketone. Darriet et al. (1995) identified the first volatile thiol, 4-mercapto-4-methyl-pentan-2-one (4MMP) in Sauvignon blanc wines in 1995. Since then, various researchers have focused on these compounds and Tominaga et al. (1996, 1998a) identified four other volatile thiols in Sauvignon blanc. 3MH and 3MHA were only recently identified in South African Chenin blanc wines (Wilson, 2017; Coetzee et al., 2018). The odour thresholds of these volatile thiols in model wine solution are 0.8 ng/L for 4MMP, 60 ng/L for 3MH and 4 ng/L for 3MHA (Tominaga et al., 1998b).

Three pathways that can lead to the formation of 4MMP and 3MH have been proposed (Roland et al., 2011). One pathway leading to the formation of 3MH includes the trans-2-hexenal and trans-2-hexenol alongside H2S, which acts as a sulphur donor. 4MMP and 3MH share the other two pathways and include cysteinylated and glutathionylated precursors. 4MMP and 3MH are synthesised in the berry and their precursors are in a cysteinylated bound form.

Tominaga et al. (1998c) identified the precursors of volatile thiols as odourless, non-volatile, non-glycosylated sulphur-CYS conjugates. These conjugates are cleaved by the yeast via its beta-lyase activity during alcoholic fermentation, and the non-volatile thiols S-3-(hexan-1-ol)-cysteine (CYS-3MH) and S-3-(4-mercapto-4-methylpentan-2-one)-cysteine (CYS-4MMP) become active compounds (Roland et al., 2010). The last pathway includes the glutathionylated precursors, such as S-3-(hexan-1-ol)-glutathione (GSH-3MH), which are released during alcoholic fermentation. 3MH precursors are located mainly in the skins, while 4MMP precursors are mostly found in the skin and pulp (Roland et al., 2011).

Only trace amounts of 4MMP and 3MH are found in grapes and musts (Dubourdieu et al., 2006). During alcoholic fermentation, these volatile thiols are released from their odourless non-volatile precursors. 3MHA is produced from the acetylation of 3MH during alcoholic fermentation by the yeast ester and forms alcohol acetyltransferase (Roland et al., 2011). Various factors, such as terroir and winemaking processes prior to alcoholic fermentation, can influence the levels of precursors present in the grapes (Murat et al., 2001b; Dubourdieu et al., 2006). Volatile thiols are susceptible to oxidation during ageing, and a decrease in levels can be expected (Coetzee, 2014). During wine ageing, 3MHA is usually converted to 3MH via acid hydrolysis (Nikolantonaki et al., 2010; Herbst-Johnstone et al., 2011) or by the breakdown of 3MH disulphide present in the wines (Capone et al., 2010; Sarrazin et al., 2010).



Grapevines have the potential to grow successfully and produce quality grapes when grown in favourable environmental conditions. The nutrition levels of a grapevine can influence the grape's composition and eventually the wine's composition and quality (Bell & Henschke, 2005). A grapevine's nutrition can be affected by various factors, such as the canopy shading (Perez-Harvey & Witting, 2001), canopy temperature (Ewart & Kliewer, 1977), cultivar (Christensen, 1984; Huang & Ough, 1989), rootstock (Christensen, 1984; Huang & Ough, 1989), season (Bell & Robson, 1999), site (Huang & Ough, 1989), soil management (Bell et al., 1979), training system (Kliewer et al., 1991), and the timing, rate and form of application of fertiliser (Bell et al., 1979; Peacock et al., 1991; Spayd et al., 1994; Christensen & Peacock, 2000; Conradie, 2001; Dufourcq et al., 2007; Lacroux et al., 2008; Jreij et al., 2009).

The nutrient requirements of a grapevine depend on its age, cultivar, yield, soil type and soil properties (Holzapfel & Treeby, 2007). Nutrition deficiencies often occur for various reasons and can be rectified by carrying out fertiliser applications.

Nitrogen supplementation is the most commonly performed soil fertilisation, as it is a primary constituent and is required by the grapevine for growth and reproduction. How much nitrogen to apply depends on the production and quality of the crops, but also on numerous research trials and years of grower experience (Christensen & Peacock, 2000). Drip irrigation is the most common way of nitrogen application, and the use of liquid materials such as aqua ammonia, urea and ammonium nitrate solution has increased, while the use of anhydrous ammonia has declined due to the high cost (Christensen & Peacock, 2000). The application rates of drip irrigation depend on vineyard conditions and this is usually applied in the spring. Research studies have reported the following foliar rates to be applied to vines: 11 to 28 kg/ha N applied to vines of medium vigour and 33 to 44 kg/ha N to below-average vines (Christensen & Peacock, 2000). Organic sources of nitrogen include farm manure, grape pomace and compost, while cover crops can also be used to add nitrogen to the soil (Christensen & Peacock, 2000).

Over the past few years, foliar fertilisation has been used widely where deficiencies or imbalances cannot be rectified by soil fertiliser applications. Foliar fertilisation is also applied as a method to improve a crop's quality and yield (Christensen & Peacock, 2000). Foliar fertiliser applications show advantages such as low cost, lack of soil fixation, being independent of root uptake, using small quantities of fertiliser, giving rise to increased quality and yield of crops, and to quick plant uptake, response and assimilation (Oosterhuis, 2009; Lasa et al., 2012). Foliar applications are preferred over soil applications when the topsoil is dry, the soil has low available nutrients, or with decreased root activity. Foliar applications are applied mostly in the case when small fertilisation corrections are to be made. The limitations of foliar fertilisation applications include leaf burn, leaf necrosis, low penetration rate, solubility problems, being washed off by rain, limited amounts can be applied at a time, and correct weather conditions are required (Watson et al., 2000). Many foliar fertilisers are soluble in water and can be applied directly to the leaves of a grapevine.

Foliar fertilisation chemicals can traverse from the leaf to the stomata via two pathways, namely an aqueous pathway and a lipoidal route (Oosterhuis, 2009). The uptake of the nutrients depends on the element's inorganic form, whether it is combined in an organic form, its ionic concentration, or the environmental conditions, which influence the time the nutrients remain in solution on the leaf (Oosterhuis, 2009). Water deficit increases the wax of the cuticle and reduces the absorption of the foliar-applied nutrient (Oosterhuis, 2009), and foliar fertilisation should therefore not be applied to grapevines experiencing a drought.

Nitrogen foliar fertilisation can be applied using urea (Lacroux et al., 2008; Dufourcq et al., 2009; Jreij et al., 2009; Wolf, 2012; Garde-Cerdán et al, 2015; Juhasz, 2015; Verdenal et al., 2015a; Garde-Cerdán et al, 2016; Geffroy et al, 2016a; Hannam et al, 2016; Verdenal et al., 2016; Dienes-Nagy et al., 2017) or amino acids (Garde-Cerdan et al., 2015). Application levels for these studies ranged from 2 to 36 kg N/ha. Sulphur foliar applications (5 and 10 kg S/ha) used elemental soluble sulphur (Dufourcq et al., 2009; Juhasz, 2015; Geffroy et al., 2016a) or micronised sulphur (Lacroux et al., 2008). Elemental sulphur is a slow-release substrate and is widely and frequently applied in the industry at various stages during the growing season of vines. Sulphur has a direct fungicidal effect and fungistatic activity on plants by increasing the plant's resistance to powdery mildew (Uncinula spp.), downy mildew and botrytis (Botrytis cinerea) (Omnia, 2015). Sulphur can also be sprayed to control mites, which can have a detrimental effect on the growth of buds and leaves during the growing season. In South Africa, the minimum sulphur content or requirement for a plant is 900 g S/kg elemental sulphur per year (Department of Agriculture, Forestry and Fisheries, 2018). A 30-day withholding period of sulphur applications is required, with 10-day intervals between applications, otherwise residual sulphur can influence the chemical composition of the grapes and can give rise to negative associated aromas from compounds such as H2S. Studies with foliar applications have not focused on applying sulphur separately to vines (Lacroux et al., 2008; Dufourcq et al., 2009). A synergy between nitrogen and sulphur results in interactions between the pathways (Téa et al., 2003; Lacroux et al, 2008). The timing of foliar sprays is critical, and nitrogen and sulphur can be applied at different stages, such as during véraison (Dufourcq et al., 2009; Jreij et al., 2009; Garde-Cerdán et al, 2015; Juhasz, 2015; Verdenal et al, 2015a; Garde-Cerdán et al, 2016; Geffroy et al., 2016a; Hannam et al., 2016; Verdenal et al, 2016; Dienes-Nagy et al, 2017), before véraison (Lacroux et al, 2008) or at the flowering stage (Wolf, 2012; Verdenal et al, 2015a).

Foliar fertilisation studies on nitrogen and sulphur applications cover a wide range of red and white cultivars and were performed in various places, such as Canada, Chile, France, Spain, South Africa, Switzerland and Virginia, USA. White cultivars included Chardonnay (Dienes-Nagy et al, 2017), Chasselas (Verdenal et al, 2015a, 2015b, 2016; Dienes-Nagy et al, 2017; Koestel et al., 2017), Colombard (Dufourcq et al, 2005, 2009; Geffroy et al, 2016a, 2016b), Gewürztraminer (Dienes-Nagy et al, 2017), Gros Manseng (Dufourcq et al, 2007, 2009; Geffroy et al, 2016a, 2016b), Melon (Dufourcq et al,; Geffroy et al, 2016a, 2016b), Négrette (Dufourcq et al, 2005, 2009; Geffroy et al, 2016a, 2016b), Sauvignon blanc (Dufourcq et al., 2011; Lacroux et al, 2008; Dufourcq et al, 2009; Jreij et al, 2009; Geffroy et al, 2010; Lasa et al, 2012; Wolf, 2012; Helwi et al, 2014; Juhasz, 2015; Geffroy et al, 2016a, 2016b; Dienes-Nagy et al, 2017; Helwi et al, 2017) and Viognier (Hannam et al., 2014). Red cultivars such as Cabernet Sauvignon (Hannam et al., 2014; Gutiérrez-Gamboa et al., 2017a), Carignan (Geffroy et al, 2012, 2016a, 2016b), Duras (Dufourcq et al, 2005), Fer Servadou (Geffroy et al, 2012, 2016a, 2016b), Malbec (Dufourcq et al., 2005), Merlot (Lasa et al., 2012; Wolf, 2012; Hannam et al, 2014, 2016), Petit Manseng (Wolf, 2012; Kelly, 2013; Kelly et al, 2017), Pinot Gris (Hannam et al, 2014, 2016), Pinot Noir (Hannam et al, 2014) and Tempranillo (Garde-Cerdan et al, 2015) were included. Only soil nitrogen fertilisation studies have been performed on Chenin blanc (Conradie, 1981; Conradie & Saayman, 1989). Foliar nitrogen fertilisation applications do not only influence the concentrations and composition of nitrogen present in the grape berry, but also indirectly influence the nitrogen levels in the must, alcoholic fermentation, and the resulting wine's composition and aroma (Petering et al., 1991; Bell & Henschke, 2005).


A grapevine's response to fertiliser applications depends on various factors, such as the genetic makeup of the cultivar, canopy management and the terroir. The chemical analysis of vine leaves, sap and soil can indicate the nitrogen content of a grapevine and can affect how the vine reacts to an application of foliar nitrogen (Bell et al., 1979; Kliewer et al., 1991; Spayd et al, 1994; Bell & Robson, 1999; Conradie, 2001; Lacroux et al, 2008; Kelly, 2013; Hannam et al, 2014; Kelly et al, 2017). The nitrogen requirement for every wine cultivar is different; for instance, the shoots, leaves and fruit of Chenin blanc accumulated up to 27 kg N/ha after soil application, and up to 60% of nitrogen reserves originate from nitrogen absorbed post-harvest (Conradie, 1986). Nitrogen-deficient grapevines (low YAN) that received nitrogen resulted in increased vine nitrogen status, which led to nitrogen metabolism and carbohydrate accumulation (Bell & Henschke, 2005).

Increased levels of nitrogen in the roots, canes and trunk increase the grapevine's ability to store nitrogen and carbohydrates (Kliewer et al, 1991; Bell & Robson, 1999). Therefore, with increased growth of the roots, the uptake of nutrients, water and nitrogen is increased (Kliewer & Cook, 1971). By applying small additions of nitrogen to a vineyard with adequate nitrogen levels, no increases in the growth and yield will happen (Kliewer et al., 1991). High vine nitrogen status may interrupt the grapevines' natural balance, causing them to become excessively vegetative and maintain vegetative growth at the expense of yield (Kliewer & Cook, 1971). It is very important that the source and sink ratio are balanced in vines, otherwise competition can occur between the sinks for carbohydrates, and grapes can have reduced composition and quality (Ewart & Kliewer, 1977). When a canopy changes due to increased growth, the microclimate in the canopy, including factors such as light, temperature, radiation, wind and humidity, are altered.

Only a few foliar fertilisation studies have focused on studying the effect of the applications on the grapevine. Most research studies on fertilisation have made use of urea, an inorganic form of nitrogen, because of the easy control of application. Factors such as the canopy nitrogen status, interior leaves, clusters, leaf layers, leaf area, pruning mass, bunch weight, yield and bunches per vine in the vineyard during ripening were studied. Kelly et al. (2017) reported that the petioles had relatively low nitrogen levels prior to fertilisation and found no differences in the number of interior leaves or clusters among fertilised treatments (15 kg/ ha urea and 15 kg/ha urea with 5 kg/ha micronised sulphur (Microthiol Disperss), applied twice before véraison), but the control had deficient YAN levels and had a greater percentage of leaf layers compared to the vines with fertiliser applications.

Another study, in which 15 kg/ha urea were applied thrice to Petit Manseng, showed no impact on the primary or secondary leaf area; however, the pruning mass per vine increased significantly compared to the control (Helwi et al., 2014). An alternative study, using foliar urea applications at 1% and 2% w/v rates applied on Merlot two weeks before véraison, at véraison and two weeks after véraison, resulted in no difference in the grapevine nitrogen status, but an increase in canopy density occurred in the second year of the experiment (Hannam et al., 2014). Lacroux et al. (2008) reported that the plots in their study were nitrogen deficient (low initial N-tester values) before performing nitrogen and nitrogen with sulphur foliar applications twice before véraison on Sauvignon blanc vines. Higher levels compared to the initial nitrogen levels were observed with the N-tester (leaf blade nitrogen content) analysis for both foliar applications, as well as a 60% increase in YAN levels (Lacroux et al., 2008).

The total yield has been found to increase upon the application of nitrogen to low nitrogen-containing vines (Bell et al., 1979; Kliewer et al., 1991; Spayd et al., 1994; Bell & Robson, 1999; Conradie, 2001; Hannam et al., 2014), while other studies have found no increases in the yield (Lacroux et al., 2008; Kelly, 2013; Kelly et al., 2017). This can be due to the initial nitrogen levels present in the vines prior to foliar fertiliser applications. Lacroux et al. (2008) also found no significant differences in bunch weight, yield, bunches per vine and pruning weight, and this can be due to the late application of nitrogen in the season. A study using a 2% w/v (28 to 36 kg N/ha/year) urea application found an increased yield of Pinot Gris, an increased number of clusters/vine for Merlot (plot 2) and Viognier, while another Merlot plot and more Pinot Gris vineyards had an increased number of clusters/vine in the following season (Hannam et al., 2014) The variable yields obtained can be due to the optimal or suboptimal timing of applications, fertiliser materials, dosage, and climatic and environmental conditions (Oosterhuis, 2009).


During the ripening period of the grape berry, the composition, colour, flavour, size, texture and susceptibility to pathogens change (Gomes et al., 2013). Tartaric acid concentrations increase during the first stages of ripening and usually remain constant after véraison. Compounds such as amino acids, micronutrients and volatile aroma compounds accumulate during the first phase of berry growth, affecting the composition of the grape berry and eventually the composition of the resulting wine (Gomes et al., 2013). During véraison, the grape berry undergoes dramatic changes and transforms from small, acidic and hard berries with very little sugar to softer, sweeter, larger, less acidic, and strongly flavoured and coloured berries. Aromatic compounds such as methoxypyrazines, which are produced during the first stage, decline during the ripening of the grape berries (Gomes et al., 2013). This can be due to the increase in sunlight and temperature levels in the cluster, and leaf-removal practices (Suklje et al., 2016).

During ripening, the grape berries rely on carbohydrates produced from photosynthesis for their development and growth. Most of the accumulation of fructose and glucose occurs after véraison. The physiological ripeness of grape berries is reached when the grapes achieve sufficient sugar levels without losing too much acidity. The phenolic and aromatic content of the berries should also be taken into consideration (Gomes et al., 2013). For a winemaker, optimal grape maturity is essential for successful alcoholic fermentation and for producing quality wines. Generally, canopies with low light produce grapes with reduced concentrations of soluble solids, total phenols and anthocyanins, while the levels of pH, TA, methoxypyrazines and potassium in the grapes increases (Kliewer, 1968; Ewart & Kliewer, 1977; Suklje et al., 2016).

Foliar fertilisation research studies have analysed °B, TA, pH, berry composition, total nitrogen content, berry volume, YAN, amino acids, ammonium, IBMP, and susceptibility to pathogens in the grapes during grape berry ripening and at harvest (Lacroux et al., 2008; Jreij et al., 2009; Mundy et al., 2009; Hannam et al., 2014; Dienes-Nagy et al., 2017; Helwi et al., 2017; Kelly et al., 2017). No foliar fertilisation studies focused on all the methoxypyrazine compounds, except on IBMP, or on the composition of separate amino acids during ripening or at harvest.

Both 1% (14 to 18 kg N/ha/year) and 2% w/v (28 to 36 kg N/ha/year) foliar urea applications increased the soluble solids at harvest for Merlot and Pinot Gris in 2010, but the 2% urea treatment resulted in a reduction in soluble solids in Merlot in the two following seasons, viz. 2011 and 2012 (Hannam et al., 2014). The 2% urea treatment caused increases in the pH levels in Merlot and Pinot Gris in 2010, but only in Pinot Gris in 2012. For all three vintages, TA levels were reduced in the grape juice of Pinot Gris with the 2% urea foliar application (Hannam et al., 2014). Similarly, Kelly et al. (2017) also found increased pH levels with foliar urea (15 kg N/ha twice before véraison) and urea with micronised sulphur (15 kg N/ha with 5 kg S/ha twice before véraison) treatments in 2011 compared to the control, while no difference was observed in the °B and TA levels (Kelly et al., 2017). Lacroux et al. (2008) found no differences in the grape composition at the harvest date for the oenological parameters of Sauvignon blanc.

During ripening, the total nitrogen content of the grapes increases, but the levels can plateau after an initial increase and, in some cases, even decline towards the end of ripening (Kliewer & Cook, 1971; Bell, 1994). Jreij et al. (2009) showed that a soil application of 60 kg N/ha ammonium nitrate (one week after bud break) with a 5 kg N/ha foliar application (at véraison) resulted in an 11% increase in berry volume compared to the control and the 30/2.6 kg N/ha treatment. The 60/5 kg N/ha treatment also showed an 11% increase in total berry nitrogen at the maturity stage compared to the control and other treatment (Jreij et al., 2009).

Hannam et al. (2014) reported that foliar applications of 2% urea solution (28 to 36 kg N/ha/year) increased the YAN levels of grape juice at harvest in almost all the vineyards in all three vintages of their study. These YAN levels were more than double compared to the control (Hannam et al., 2014). YAN levels were increased by 60% with applications of foliar nitrogen (10 kg N/ha urea, two applications prior to véraison) and foliar nitrogen (10 kg N/ha urea twice before véraison) with sulphur (5 kg S/ha micronised sulphur twice before véraison) compared to the control (Lacroux et al., 2008). Dienes-Nagy et al. (2017) reported that YAN levels increased proportionally with foliar urea applications compared to the control over three vintages. This shows that vines are able to redistribute the nitrogen that is absorbed from the leaves to the grape berry.

The total amino acid concentration has been found to increase from véraison to harvest (Kliewer, 1968; Bell, 1994), while in other cases the concentration stabilised and declined after véraison (Kliewer, 1968). During grape berry ripening, the ammonium concentration of the grape berries declines (Bell, 1994). Foliar urea applied to white cultivars resulted in an increase in primary and secondary amino acids, but a decrease in ammonium nitrogen levels was observed during grape berry ripening compared to the control (Dienes-Nagy et al., 2017). Bell (1994) reported that PRO accumulation increases during grape berry ripening, while some researchers have reported PRO levels to stabilise and slowly decline until harvest (Bell & Henschke, 2005). PRO generally accumulates due to abiotic stresses such as salt stress, cold stress, water stress and heat stress (Kavi Kishor & Sreenivasulu, 2014). ARG increased from véraison and reached a maximum at and started to decline during ripening (Kliewer, 1968; Bell, 1994), while other studies found that ARG continued to increase until harvest.

The susceptibility of grapes to the fungal pathogen Botrytis cinerea can be enhanced by a high content of foliar urea applications, although Lacroux et al. (2008) found contradictory results that showed no increase in the incidence of Botrytis. A strong positive correlation between sugar concentration and susceptibility to Botrytis cinerea in intact grape berries of Sauvignon blanc has been found, and the wounding of unripe berries with low sugar can also increase the susceptibility to the fungal pathogen (Mundy et al., 2009).

Grape must to wine

After grapes are harvested, some grape compounds can be subject to degradation due to biological, chemical or physical processes. These processes can influence the composition and quality of the grapes and also of the resulting wine. The nitrogen composition of grape berries at harvest, including the total nitrogen content, primary and secondary amino acids, ammonium and thiol precursors, can be influenced positively by nitrogen fertilisation (Conradie, 2001; Chone et al., 2006). Mechanical machine harvesting can activate a variety of processes, such as oxidation, thereby releasing the precursors of volatile thiols or microbial metabolism. Harvested white grapes usually undergo crushing and destemming, and SO2 is added to prevent oxidation. Thereafter, the grape undergoes a period of skin contact, after which it is pressed. During alcoholic fermentation, the grape must is transformed into wine by the yeast. In other words, the high sugar concentration of glucose and fructose is converted by various metabolic steps, leading to the production of CO2 and ethanol. The yeast growth depends on the available nutrients, such as nitrogen provided by the grapes (Henschke & Jiranek, 1993). When the must is deficient in nitrogen content (< 140 mg N/L, low YAN), the winemaker can adjust the nitrogen levels by adding a source of nitrogen, usually DAP. During alcoholic fermentation, a large number of aroma compounds such as volatile thiols and major volatiles are released from their precursors (Lambrechts & Pretorius, 2000; Swiegers et al., 2005) and contribute to the 'fermentation bouquet' (Bell & Henschke, 2005). The maturation of wine in the bottle or in an oak barrel can influence the chemical compound makeup and aromatic expression (Botha, 2015).

Research studies on foliar and soil fertilisation have focused on measuring °B, alcohol, CYS-3MH, FAN, GSH, GSH-3MH, malic acid, pH, TA, tartaric acid, YAN, amino acids, esters, methoxypyrazines, higher alcohols and volatile thiols in the must, juice, fermentation juice and/or resulting wines (Conradie, 2001; Chone et al., 2006; Lacroux et al., 2008; Dufourcq et al., 2009; Lasa et al., 2012; Helwi et al., 2014; Geffroy et al., 2016a; Dienes-Nagy et al., 2017; Gutiérrez-Gamboa et al., 2017b).

Oenological parameters, such as °B, alcohol, pH, TA, tartaric acid and malic acid, have been measured and no differences were found in the composition of the grape juice and must between the treatments (Lacroux et al., 2008). Lacroux et al. (2008) reported that, with Sauvignon blanc, the soil ammonium nitrate application (30 kg N/ha) resulted in higher alcohol levels in the wines compared to the control and foliar fertilisation applications. Foliar applications did not significantly affect the oenological parameters of Tempranillo and Monastrell grape juice (Garde-Cerdan et al., 2015), but late foliar applications of urea (15 days after véraison) increased the acidity of wines made with Sauvignon blanc and Merlot (Lasa et al., 2012).

GSH in musts and wine plays a crucial role by protecting varietal volatile thiols from oxidation, and therefore protects the aroma production of Sauvignon blanc wines (Lavigne et al., 2007). The GSH levels in wines were higher for applications of foliar urea (10 kg N/ha twice before véraison), and foliar urea (10 kg N/ha twice before véraison) with sulphur (5 kg S/ha twice before véraison), compared to the control (nitrogen deficient) and soil fertilisation (Lacroux et al., 2008). The urea with sulphur application did not increase the GSH levels compared to the urea treatment (Lacroux et al., 2008). Foliar urea and foliar urea with sulphur applied at véraison and two weeks later did not show significant effects on the GSH levels in the musts compared to the control (Gutiérrez-Gamboa et al., 2017a). Soil nitrogen fertilisation of 60 kg N/ha at berry set to Sauvignon blanc showed a 670% increase in GSH compared to the control (Chone et al., 2006).

Helwi et al. (2014) applied soil fertilisation (50 kg N/ha, two applications), soil (100 kg N/ha, two applications) and foliar fertilisation of urea (15 kg N/ha, three applications) to Sauvignon blanc in two locations. The glutathionylated precursor of 3MH (PGSH-3MH) increased during ripening for all the treatments, and with soil and foliar treatments the levels were higher compared to the control. The results were highly correlated with the nitrogen status of the grapes. The cysteinylated precursor of 3MH (PCYS-3MH) was stable in the berries from one of the Sauvignon blanc plots during berry development, but increased with nitrogen foliar application at mid-maturity and decreased thereafter (during maturity) for all the treatments. Conversely, on the other Sauvignon blanc plot, the PCYS-3MH concentration increased during ripening and was higher in the soil and foliar nitrogen applications compared to the control (Helwi et al., 2014).

The nitrogen content ofthe must was increasedby 50% and 100% respectively with 10 and 20 kg N/ha foliar applications to white cultivars (Sauvignon blanc) (Geffroy et al., 2016a). Dufourcq et al. (2009) reported significant increases in berry YAN with urea foliar fertilisation at véraison. Nitrogen soil applications resulted in a 10% decrease in amino acids in relation to YAN (Spayd et al., 1994). Foliar urea treatments on white cultivars, such as Chasselas, Sauvignon blanc and Chardonnay, showed a correlation between YAN and PRO levels in the wines (Dienes-Nagy et al., 2017). The amino acid content of a vine can be influenced and is dependent on various factors, such as the cultivar and rootstock genetics, climatic conditions, terroir, nitrogen content, soil, and canopy management practices (Conradie, 2001; Bell & Henschke, 2005; Lee & Schreiner, 2010). Increases of 44% and 75% in ARG and ALA respectively were obtained from urea with sulphur applications at véraison and two weeks later (Gutiérrez-Gamboa et al., 2017a). This study also showed that, by comparing applications of foliar urea to foliar urea with sulphur, the latter application resulted in an improved amino acid content (Gutiérrez-Gamboa et al., 2017a). Lasa et al. (2012) showed that, with foliar applications of urea, the accumulation and synthesis of amino acids such as ARG, GLN, THR and ALA were significantly increased. This gives rise to better alcoholic fermentation kinetics and the higher production of ethyl esters, which are essential for quality wines (Lasa et al., 2012).

Methoxypyrazine levels in the must can determine the levels in the resulting wine. Helwi et al. (2017) reported that nitrogen soil fertilisation did not significantly affect the IBMP levels in Sauvignon blanc berries, wine or must compared to the control (deficient YAN). IBMP levels were slightly higher in the resulting wine than in the must (3 to 5 ng/L vs 0.5 to 1 ng/L) (Helwi et al., 2017). Another study reported that a high level of irrigation with additional nitrogen (60 kg/ ha at fruit set) promoted canopy growth, with higher levels of IBMP during fruit maturation, in Merlot vines (Mendez-Costabel et al., 2014). No foliar fertilisation research studies have focused on the impact of foliar nitrogen fertilisation on the methoxypyrazine content, such as the IBMP, IPMP and SBMP levels. Researchers speculate that foliar fertilisation with urea can increase these levels in the grapes and consequently in the resulting wine.

Various wines produced for nitrogen fertilisation studies have resulted in lower concentrations of higher alcohols. Higher alcohols are directly related to amino acid metabolism and are directly influenced by the YAN levels. Higher alcohols reach a peak between 200 and 300 mg N/L YAN, and degrade if the YAN levels increase (Ugliano et al., 2007). Increased levels of esters are found mostly with nitrogen fertilisation applications (Lasa et al., 2012). In this case, wines are described as being 'fruitier' compared to the control. Fatty acids, ethyl esters and acetates contribute to the fruity aroma of white wines, and their levels increase with DAP additions and higher YAN levels in the must (Ugliano et al., 2007).

In vineyards containing low (< 140 mg N/L) and moderate nitrogen (140 to 300 mg N/L) (Gardner, 2014) to which nitrogen fertiliser applications were applied, there was an increase in YAN, 3MH, 3MHA and 4MMP levels, which were much higher compared to the control (Peyrot des Gachons et al., 2005; Chone et al., 2006). 4MMP was at higher levels after the foliar urea application (10 kg N/ha twice before véraison) compared to the control for Sauvignon blanc, while 3MHA and 3MH levels were not higher (Lacroux et al., 2008). The application of foliar urea (10 kg N/ha twice before véraison) with sulphur (5 kg S/ha twice before véraison) resulted in higher concentrations of 3MH, 3MHA and 4MMP compared to the control (Lacroux et al., 2008). Geffroy et al. (2016a) also reported that 10 and 20 kg N/ha foliar applications with 5 and 10 kg S/ha respectively resulted in musts with a three- to four-fold gain in varietal thiols in the case of white cultivars, including Sauvignon blanc. Dufourcq et al. (2009) reported significant increases in berry YAN with urea foliar fertilisation at véraison, and these musts correlated with higher 3MH and 3MHA concentrations in the resulting wine. Helwi et al. (2014) applied soil fertilisation (50 kg N/ha - two applications and 100 kg N/ha - two applications) and foliar fertilisation of urea (15 kg/ha - three applications) to Sauvignon blanc and reported that all the wines produced from vines with higher nitrogen status contained more 3MH.

Wines produced from grapevines that received applications of soil ammonium nitrate (30 kg/ha), foliar urea (10 kg N/ha, twice before véraison), and foliar urea (10 kg N/ha, twice before véraison) with sulphur (5 kg S/ha, twice before véraison), underwent sensory evaluation (Lacroux et al., 2008; Geffroy et al., 2016a). The wines that received foliar urea with sulphur applications had significantly higher aroma intensity, while wines that received foliar urea had decreased intensities (Lacroux et al., 2008). Geffroy et al. (2016a) also reported that the foliar applications of 10 and 20 kg/ha N and 5 and 10 kg/ha S resulted in wines with more intense and increased notes of 'grapefruit' and 'tropical fruit', while no undesirable sulphur-related notes were perceived.



A wine's flavour and aroma expression are influenced by various reactions and interactions of chemical compounds (Fischer, 2007). These non-volatile and volatile compounds can be derived from the grape, produced from the yeast metabolism during alcoholic or malolactic fermentation, or produced during barrel or bottle ageing (Fischer, 2007).

Various foliar fertilisation studies show that nitrogen and nitrogen with sulphur applications can positively affect various volatile and non-volatile compounds in grapes, musts and the resulting wines. Foliar fertiliser applications of nitrogen and nitrogen with sulphur applied at véraison have shown a positive impact on berry chemical content, yeast growth and metabolism, and produce more aromatic wines. By undertaking nitrogen and sulphur foliar applications at véraison, the concentrations of sulphur- and nitrogen-containing compounds can be increased. Methoxypyrazines and major volatiles are important volatile compounds and have not been analysed in foliar fertilisation research studies. Only a few research studies have determined vine nitrogen status before and after foliar applications (Lacroux et al., 2008; Jreij et al., 2009; Helwi et al., 2014). Only two studies have undertaken sensory analysis of the finished wines, but these do not mention the age of the wines (Lacroux et al., 2008; Geffroy et al., 2016a). Another void in research studies on foliar fertilisation is that the chemical and sensory evolution of wines during bottle maturation has not been considered. The information gained by performing sensory analysis during different stages of bottle maturation can add to the knowledge base of the aroma of these wines.

Most foliar studies have mentioned and proven that they have found positive correlations where fertiliser applications were done prior to and at véraison, due to the vine's nutrient uptake patterns and requirements. Many research studies have been done on the biochemical and chemical origins of non-volatile and aroma compounds, and the effects of the environment, and viticultural and winemaking practices on the concentrations on these compounds. By performing chemical and sensory analysis to understand the influence of the effect of foliar fertilisation with nitrogen and nitrogen with sulphur on the final wines, the wine industry can make decisions at the viticultural and winemaking level to produce wines with more desirable sensory attributes.



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Submitted for publication: October 2018
Accepted for publication: February 2019



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Acknowledgements: The authors would like to thank Winetech for financial support

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