Olivier GEFFROY Associate professor in & enology [email protected]

Rotundone in red

An overview of the impact of plant material, environmental factors and viticultural techniques

4th Annual IVES - OENO One SCIENCE MEETING February 6, 2020 - Maison Hennessy, Cognac A few introductive words on aroma

➢More than 800 volatile compounds ➢About 10% of them have a sensory impact

➢Odorants belong to several chemical families thiol ➢Concentration levels from ng/L to hundreds of mg/L Ex. 3-sulfanylhexanol ➢If the varietal aroma of white wines is quite easy to apprehend (monoterpenols, varietal thiols…) ➢Those of red wine remained more mysterious or limited to undesirable molecule such as IBMP…. ➢Until the discovery of rotundone in 2008 ! Alkyl-méthoxypyrazines Ex. IBMP

2 Arôme poivré des vins rouges Interesting facts about rotundone (1/2)

➢Sesquiterpènes (C15H22O) = active compounds of essential oils ➢Found in Cyperus rotundus, pepper and aromatic plants ➢Odour threshold of 8 ng/L (water) and 16 ng/L (wine) ➢Heavy compound less volatile than monoterpenes ➢Produced by enzymatic oxydation of α-guaiene ➢20-25% of specific anosmia reported ➢Positively perceived except by young wine consumers ➢Particularly appreciated by wine connoisseurs who usually spent more than 10 euros for a bottle wine Wood et al. (2008), JAFC

3 Arôme poivré des vins rouges Interesting facts about rotundone (2/2)

96-99% 4-1% ➢96-99% of rotundone is located in the skin ➢Hydrophobic compound (≈10% extracted) ➢Strong affinity with particles ➢Winemaking practices have been identified to lower rotundone in wine (Saccharomyces uvarum, carbonic maceration)… ➢But not to enhance rotundone, including the increase in maceration temperature and the use of pectolytic enzyme ➢Effort to maximize rotundone in wine must be undertaken in vineyard !

Caputi et al. (2011), JAFC

4 Impact of genotypeArôme poivré des vins rouges

Occurrence in wines frequently described as peppery (2008-2015)

* Tardif (n=2) Manseng noir (n=2) (n=2) * >50 ng/L *Prunelard (n=1) *Négrette (n=1) * (n=1) * >16 ng/L * (n=2) (n = 15) South West of France * * (n=1) * (n=1) *Pineau d'Aunis (n=3) *Mourvèdre (n=1) * Mondeuse (n=2) OtherFrance * (n=3) * Maturana tinta (n=1) Touriga nacional (n=1) Graciano (n=2)

Foreign * * Arani (n=1) Geffroy (2018), Journée R&D Purpan 0 50 100 150 Geffroy (2018) 5 Impact of genotypeArôme poivré des vins rouges

Occurrence in wines frequently described as peppery (2008-2015)

* Tardif (n=2) Manseng noir (n=2) Tannat (n=2) * >50 ng/L *Prunelard (n=1) *Négrette (n=1) * Malbec (n=1) * >16 ng/L * Fer (n=2) Duras (n = 15) South West of France * * Abouriou (n=1) *Pinot noir (n=1) *Pineau d'Aunis (n=3) 19-62 ng/kg *Mourvèdre (n=1) * Mondeuse (n=2) OtherFrance * Gamay (n=3) 243 ng/kg * Maturana tinta (n=1) Touriga nacional (n=1) Graciano (n=2) 21 ng/kg

Foreign * * Arani (n=1) Geffroy (2018), Journée R&D Purpan 0 50 100 150 Takase et al. (2015), AJEV Geffroy (2018) Logan (2015), Doctoral Thesis 6 Impact of genotypeArôme poivré des vins rouges

Occurrence in wines frequently described as peppery (2008-2015)

* Tardif (n=2) Manseng noir (n=2) Tannat (n=2) * >50 ng/L *Prunelard (n=1) *Négrette (n=1) * Malbec (n=1) * >16 ng/L * Fer (n=2) Duras (n = 15) South West of France * * Abouriou (n=1) *Pinot noir (n=1) Up to 1345 ng/L *Pineau d'Aunis (n=3) in spirits (whisky, Mourvèdre (n=1) * tequila, rum) * Mondeuse (n=2)

OtherFrance Gamay (n=3) * Concentrations * Maturana tinta (n=1) Touriga nacional (n=1) increase with barrel Graciano (n=2) aging time

Foreign * * Arani (n=1) Geffroy (2018), Journée R&D Purpan 0 50 100 150 Genthner (2014), Master Thesis Geffroy (2018) 7 Impact of genotypeArôme poivré des vins rouges

Clonal differences observed for Duras Geffroy et al. (2015a), JISVV ➢Clones 554 and 654 showed the greatest concentrations… ➢While clone 555 the lowest ➢In accordance with other works conducted on Shiraz (Siebert and Solomon 2011) and Grüner Veltliner (Caputi et al. 2011) ➢How to explain the extremely high levels for the 2014 vintage ?

8 Impact of biotic Arômefactors poivré des vins rouges

Powdery mildew (Erysiphe necator) Geffroy et al. (2015a), JISVV

450 ➢Identification of a positive correlation between PM severity on bunches and 400 rotundone concentration 350 ➢Rotundone could be produced in 300 response to PM infection 250 ➢Systemic effect as wines were 200 produced from healty berries ? 150 Rotundone concentration (ng/L) concentration Rotundone 0 10 20 30 40 50 60 Powdery mildew severity (%)

9 Impact of biotic Arômefactors poivré des vins rouges

Bunch rot ()

Geffroy et al. (2019a), OENO One ➢Negative correlation between rotundone 300 concentration and levels of gluconic acid 250

➢Due to the fungus or to its laccase ? 200

➢Rather unexpected as laccase has the ability to 150 convert α-guaiene into rotundone (Schilling et al., 2013) 100 ➢However, laccase can neutralize the toxic effect of 50 sesquiterpenes through oxydation (Mayer et Staples, Rotundone concentration (ng/L) concentration Rotundone 0 2002) 0 100 200 300 Gluconic acid (mg/L)

10 Impact of abioticArômefactors poivré des vins rouges

Modeling rotundone in Duras wines

PDO Gaillac ➢10 Duras blocks were monitored in 2013 and in 2014 ➢+ 50 variables were collected (fruit, vine, climate) and Ψstem was modelized ➢Rotundone was determined in wines made from microvinification techniques (1L Erlenmeyer flasks) ➢PLSR models were built to model rotundone in 2013, 2014 and in both vintages

11 Impact of abioticArômefactors poivré des vins rouges

Modeling rotundone in Duras wines Geffroy et al. (2019a), OENO One

300 ➢Some blocks with high 2013 2014 250 rotundone levels in 2013 (i.e. n°2 and 4) exhibited low levels 200 in 2014 and conversely (i.e. n°3) 150 ➢Fixed variables (topography, plant material…) do not allow to 100

explain the differences between 50

blocks (ng/L) concentration Rotundone 0 1 2 3 4 5 6 7 8 9 10 Site number

12 Impact of abioticArômefactors poivré des vins rouges

Modeling rotundone in Duras wines Geffroy et al. (2019a), OENO One

➢Positive contribution of cumulative rainfall, daily irradiation and hours of sunshine to the models ➢Negative contribution of the Huglin index ➢Water supply and light stimulate rotundone production while temperature has a depreciative effect ?

13 15 2011 14 a 2012 ab a Arôme poivré 13des vins rougesb How to manipulate rotundone12 concentration in wine ? c 11

Throught harvesting date 10 Degré Potentiel [% Vol] [% Potentiel Degré 9 Geffroy et al. (2014), AJGWR

70 ➢Kinetic of accumulation slightly 2011 60 differ between the two vintages 2012 a 50 a ➢In 2011, fast increase and then a reach a plateau 40 30 b

➢In 2012, steady increase Rotundone (ng/l) Rotundone 20 b

➢To enhance rotundone, dont pick 10 too early ! 0 50% ver.+30 50% ver.+37 50% ver.+44 50% ver.+51 50% ver.+58

14 Arôme poivré des vins rouges How to manipulate rotundone concentration in wine ?

Throught irrigation and defoliation Geffroy et al. (2014), AJGWR

70 ➢No impact of thinning 2011 +28% 60 a (40%) and jasmonic acid 2012 +28% ab 50 ab

spraying on rotundone /l) b ng

( 40 ➢Positive effect of irrigation +39% +39% (4x10mm before veraison) 30 -69% 20 -69% Rotundone c -53% ➢Depreciative effect of -53% defoliation (2 faces at mid- 10 veraison) 0 Control Irrigation Jasmonic Defoliation Grape thinning acid

15 Arôme poivré des vins rouges How to manipulate rotundone concentration in wine ?

Enhancing rotundone through irrigation while mitigating the depreciative impact on phenolics

Geffroy et al. (2016), Scientia Horticulturae ➢While enhancing rotundone accumulation, irrigation is likely to have a depreciative impact on phenolic compounds (direct and indirect) ➢Can a cutting of the fruit bearing cane (PES) 20 days before compensate this loss ? ➢We showed in 2013 and in 2014 that PES had a négligeable impact on rotundone accumulation ➢In 2014, implementation of a viticultural system combining irrigation (5x14mm) + PES

16 Arôme poivré des vins rouges How to manipulate rotundone concentration in wine ?

Enhancing rotundone through irrigation while mitigating the depreciative impact on phenolics

Parameter Control Irrigation + PES Δ / control Alcohol (% Vol.) 12.9 b 14.4 b +1.5 % vol. Total acidity (g/L tartaric acid) 5.59 b 6.51 a +0.92 g/L pH 3.60 a 3.61 a = Tartaric acid (g/L) 1.21 a 1.22 a = Volatile acidity (g/L acetic acid) 0.47 a 0.55 a = Anthocyanins (mg/L) 1061 b 1199 a +138 mg/L Total Phenol Index (TPI) 69.6 b 85.0 a +15.4 points (kg/vine) 2.12 a 1.78 b -16% Rotundone (ng/L) 30 b 44 a +47%

Geffroy et al. (2016), Scientia Horticulturae

17 70 A 60 Arôme poivré des vins rouges 50 How to manipulate rotundone concentration in40 wine ? 30

20 Throught differential harvest 10

r² = 0.76 rotundone concentration rotundone (ng/L) 0 Geffroy et al. (2014), AJGWR -27.20 -27.00 -26.80 -26.60 -26.40 -26.20 -26.00 -25.80

δ13C

➢Correlation between 70 40 A B rotundone and δ13C 60 35 30 50 ➢Rotundone is sensitive to weak 25 40 variation of water constraint 20 30 15 20 ➢Possibility to organize 10

10 differential harvest in order to r² = 0.76 5

rotundone concentration rotundone (ng/L) r² = 0.74 produce wines with distinct 0 (ng/L) concentration rotundone 0 -27.20 -27.00 -26.80 -26.60 -26.40 -26.20 -26.00 -25.80 -25.50 -25.00 -24.50 -24.00 -23.50 -23.00 -22.50 levels of peppery intensity ? δ13C δ13C 40 B 35

18 30 25

20

15

10

5 r² = 0.74 rotundone concentration (ng/L) concentration rotundone 0 -25.50 -25.00 -24.50 -24.00 -23.50 -23.00 -22.50 δ13C Arôme poivré des vins rouges How to manipulate rotundone concentration in wine ?

Throught differential harvest Geffroy et al. (2015b), GiESCO meeting

➢We set up a methodology based on stratified sampling to get access to rotundone spatial distribution ➢25 measurements of Trunk Circumference (TC) on a 0.40- ha block ➢Positionning of 6 smart points each one composed of 50 vine plants ➢For each smart point, we monitored fruit and plant variables

19 Arôme poivré des vins rouges How to manipulate rotundone concentration in wine ?

Throught differential harvest Geffroy et al. (2015b), GiESCO meeting

8 ➢Average TC class has more similarity Nb of clusters Yield 6 with the weak TC class and these two NBI (closure) classes should be merged 4 NBI (harvest) C ➢Rotundone is well correlated with δ13C TA (harvest) Nb shoots 2 Weight 200 berries E YAN (harvest) at veraison(r2 =0.93) and harvest (r2 = PWW (harvest) 0.82), Ψstem (harvest) Rotundone NBI (berry set) Ψstem (closing) 0 -6 -4 -2 0 2 4 NBI (veraison) 6 BC A Ψstem (50% ver +25) C D δ13C (veraison) δ13C (harvest) ➢94.5 ng/L (large TC), 60.5 and 61 ng/L (18,26%) PC2 Malic (harvest) Malic (veraison) -2 (average and weak TC) Brix (harvest) Anthocyanins (harvest) F -4 δ13C (closing) ➢TC can be used to map rotundone Ψstem (50% ver) NBI (berry set +10) TPI (harvest)

-6 PC1 (59,97 %) 20 Arôme poivré des vins rouges How to manipulate rotundone concentration in wine ?

Enhancing rotundone in Fer wines while decreasing IBMP Geffroy et al. (2019b), OENO One

➢Fer represents ≈ 1500 ha in the SW of France 4 3.5 a ➢Within the PDO Marcillac, Fer wines exhibit 3 intense green and peppery notes imputable to 2.5 IBMP and rotundone 2 b b 1.5 c ➢Leaf removal, removal of lateral shoots and 1 delayed harvest can be used to modulate the 0.5

aroma of Fer wines towards an increase in IBMP of /OAV rotundone of OAV 0 Control Leaf removal Removal of Delayed lateral shoots harvest OAVrotundone / OAVIBMP ratio

21 Arôme poivré des vins rouges How to manipulate rotundone concentration in wine ?

Enhancing rotundone in Fer wines while decreasing IBMP Geffroy et al. (2019b), OENO One

➢Fer represents ≈ 1500 ha in the SW of France 4 3.5 a ➢Within the PDO Marcillac, Fer wines exhibit 3 intense green and peppery notes imputable to 2.5 IBMP and rotundone 2 b b 1.5 c ➢Leaf removal, removal of lateral shoots and 1 delayed harvest can be used to modulate the 0.5

aroma of Fer wines towards an increase in IBMP of /OAV rotundone of OAV 0 Control Leaf removal Removal of Delayed lateral shoots harvest OAVrotundone / OAVIBMP ratio

These results might be transposable to Cabernet Franc ?

22 Arôme poivré des vins rouges How to manipulate rotundone concentration in wine ?

Enhancing rotundone in Fer wines while decreasing IBMP Geffroy et al. (2019b), OENO One 80 16 70 2015 2016 14 a ab 60 ab a 12 50 b b 10 40 b 8 30 b 6 20 (ng/L) IBMP Rotundone (ng/L) Rotundone 4 10 2 0 Control Leaf removal Removal of lateral Delayed harvest 0 shoots Control Leaf removal Removal of Delayed lateral shoots harvest ➢Significant decrease in IBMP observed for removal of lateral shoots and delayed harvest ➢ Highest rotundone levels found for delayed harvest while levels for leaf removal were intermediate 23 Arôme poivré des vins rouges Take home message

➢Rotundone is a positive aroma compound ➢It is a rather ubiquitous molecule ➢Certified clones have an impact on rotundone concentration in wine ➢The production of rotundone by the plant could be a response to biotic stress (i.e. PM) ➢Botrytis cinerea has a negative impact on rotundone in wine ➢Rotundone accumulation is also affected by abiotic factors (↗with water supply and light, ↘ with temperature) ➢The compound is not impacted by grape thinning and crop load ➢Harvesting date, irrigation (+PES), defoliation and selective harvest are leverages to manipulate rotundone levels in wines

24 Arôme poivré des vins rouges Thanks to your attention and to the partners Technical partners

Funding partners

25 Arôme poivré des vins rouges What did I bring for tasting ?

Sample n°1 Sample n°2 Sample n°3

200 ng/L ≈ 60 ng/L ≈ 20 ng/L

Gaillac PDO Gaillac PDO Rotundone 100% Duras 100% Duras solution Intense defoliation No defoliation Aged in 2nd hand barrels 26