Opticl Properties of Berry Epicuticulr Wxes in Four Georgin Grpe ultivrs ( Vitis vinifrr L.) L. Rustioni 1, D. Mghrdz&, 0. Fill 1 Universitâ degli Studi di Milno, IRJVE - entro Interdiprtimentle di Ricerc per l'innovzione in Viticoltur ed Enologi, vi elori 2, 1-20133 Milno, Itly Institute of Horticulture, Viticulture nd Oenology, 6 Mrshl Gelovni Ave., 0159 Thilisi, Georgi Submitted for publiction: November 2011 Accepted for publiction: My 2012 Key words: Epicuticulr wxes, reflectnce, berry physiology, visible, infrred The epidermis of plnt shoot orgns is generlly covered by epicuticulr wxes. The role of this lyer is relted minly to the protection of the inner tissues from biotic nd biotic stresses. In this study, ttention is focused on the opticl properties of the berry epicuticulr wxes of the grpevine (Vids vinjfer L.) t fruit ripening. The reflectnce of the berry surfce ws mesured before nd fter chloroform tretments in four Georgin grpe cultivrs grown in northern Itly. Epicrp opticl properties were underlined t different wvelengths (341 to 1025 nm) for ech cultivr. The results show tht the berry wxes hve severl opticl properties; it is possible tht their min eco-physiologicl effect is to provide specific protection ginst different types of rdition. Besides the physiologicl spects, the results re interesting lso from technicl point of view in the field of interprettion of reflectnce mesurements crried out by non-invsive instruments. INTRODUTION Epicuticulr wxes on most plnt surfces (generlly fruits nd leves) re deposited s smooth, trnsprent lyers. However, surfce wxes on mny plnts crystllise into structures tht look like coting with whitish-bluish colour. Plnts hving this feture re sid to be glucous or to hve wx bloom. When glucous surfces re exmined using scnning electron microscopy, myrid of unique epicuticulr wx crystls, specific to vrious species nd their orgns, re observed (Jenks & Ashworth, 1999). An observtion of the fine structure underlines tht the most common structurl model of the mtrix portion of the cuticle is tht of bilyered cuticulr membrne. The outer portion of the cuticle is encrusted with wxes (intrcuticulr wxes), nd thin film of epicuticulr wxes covers the outer surfce. In certin species, more or less dense cover of epicuticulr crystls my occur nd, becuse they sctter light, they re responsible for the distinctive visul ppernce (Rosenquist & Morrison, 1988; Muller & Reiderer, 2005). In chemicl sense, wxes re esters of long-chin ftty cids with long-chin primry lcohols. In brod sense, plnt epicuticulr wxes include mixture of hydrophobic compounds, with other lipoidl molecules found in the wxes themselves. The structure nd chemistry of wxes cn differ mong plnt species, ecotypes of the sme species, nd orgns of the sme plnts. They cn lso vry during plnt development, nd differences hve lso been observed on different prts of the sme lef (Jenks & Ashworth, 1999; Muller & Reiderer, 2005). The physicochemicl chrcteristics of these surfce lyers ply n importnt role in plnt resistnce to vriety of biotic nd biotic stresses, including those cused by fungl nd bcteril pthogens, phytophgous insects, drought, freezing temperture, solr rdition, mechnicl brsion, nthropogenic influences, uptke nd efficiency of plnt growth regultor, nd minerl nutrient, pesticide nd herbicide sprys (Rosenquist & Morrison, 1988, 1989; Jenks & Ashworth, 1999; Muller & Reiderer, 2005). It is common observtion tht plnts growing in environments with high levels of solr rdition often hve thicker lyer of epicuticulr wxes, which cn reflect 20 to 80% of the incoming rdition, while plnts growing in shde conditions reflect bout 10%. Even if plnt cuticles bsorb ultrviolet rdition to vrying degrees, the epicuticulr wxes of most species do not bsorb significnt mounts of rdition. However, epicuticulr wxes on some species my provide protection by scttering nd reflecting incoming rdition. The size, distribution nd orienttion of the wx crystls, nd other surfce fetures, determine the extent to which light is scttered t the tissue surfce. Generlly, rdition is scttered cross the spectrum (in extreme cses it my turn plnt surfces whitish s result of brodbnd reflection in the visible prt of the solr spectrum), but in some species there is preferentil scttering of shorter * orresponding uthor: E-mil: Lur.Rustioni@unimi.it Acknowledgements: Joint publiction of the OST Action FA1003, "Est-West ollbortion for Grpevine Diversity Explortion nd Mobiliztion of Adptive Trits for Breeding". We thnk Dr Mrin Fogrty for revising the text S. Afr. J. EnoL Vitic., VoL 33, No. 2,2012 138
139 Reflectnce of Epicuticulr Wx wvelength rdition (Jenks & Ashworth, 1999; Muller & Reiderer, 2005). Holmes nd Keiller (2002) demonstrted tht lef wxes were very effective reflectors of both UV nd longer wvelength rdition. Euclyptus lef reflectnce ws substntilly greter t ll the wvelengths scnned compred with not-wxy nd chloroform-treted leves. Suffice it to sy tht the behviour of succulent plnts ws different, underlining specfficity of the wx properties (Holmes & Keiller, 2002). In fruits, nd especilly in grpes, reserch on wx properties is generlly focused on the nti-trnspirnt properties, which re prticulrly importnt in post-hrvest mnipultions for conservtion, the drying processes (Mhmutoglu et l., 1996; Pngvhne et l., 1999; Di Mtteo et l., 2000; Doymz & Pl, 2002; Doymz, 2004, 2006; Mugnu et l., 2011), nd protection ginst pthogens such s Bolrylis cinere (Mrois et l., 1986; Rosenquist & Morrison, 1988, 1989; Percivl et l., 1993). Rosenquist nd Morrison (1988) studied the formtion, shpe nd modifiction of the epicuticulr wxes on Thompson Seedless grpes. They observed tht smll, individul, upright pltelets begin to tke shpe few dys fter nthesis. These rpidly incresed in both size nd number, reching the highest density during the lg phse. In the end the pltelets spred prt s the berry resumed rpid growth fter vérison. In ddition to growth in size, the wx pltelets lso incresed in complexity during berry development, strting s smll, simple pltes with blunt edges, nd finishing s overlpped nd lce-like pltes, teiminting in shrply lobed edges (Rosenquist & Morrison, 1988). The im of this work ws to study the opticl properties of grpe berry epicuticulr wxes in different cultivrs with vrious skin colours, mesuring the reflectnce of the surfces before nd fter wx removl by chloroform tretments. MATERIALS AND METHODS In 2011, ripe bunches of four grpe vrieties ( Vilis vinfer L.) were collected in the germplsm collection vineyrd of the Regionl Reserch Sttion of Riccgioi (Lombrdy region, northern Itly). The site is locted in the Oltrepô pvese viticulturl re (long. 9 05', lt. 44 58', elevtion 144 m.s.l.) on hilly terrce with slight est exposition with typicl cly soil (Udic Pleustlfs fine silly, mixed, superclive, mesic following the USDA soil txonomy by Soil Survey Stff, 1999). According to Koeppen's clssffiction (Strhler & Strhler, 2004), the experimentl site hs mesotherml climte with trnsitionl chrcters between n Ocenic type (fb) nd Mediterrnen one (s). The nnul verge temperture is 11.7 nd the verge temperture of Jnury nd July is 0.7 nd 22.4 respectively. The pluviometric regime presents n nnul rinfll vrying between 600 nd 800 mm, with two mxim (in spring nd fll); the winter pluviometric minimum is reflective of the Ocenic type, wheres the summer minimum is indictive of the Mediterrnen system. The vineyrd ws estblished in 2006. Plnts were spced t 2.5 m (inter-row) x 1 m (intr-row) with density of 4000 plnts/h, Guyot trined, with two-bud spur nd 10- to 12-bud cne. The inter-row soil ws kept weed free by two yerly glyphoste herbicide tretments. The plnt propgtion mteril ws tken from the grpevine collection ofgeorgin ncient cultivrs estblished in Dighomi (Thilisi, Georgi) in 1967/1968. Ech ccession ws represented by five plnts. The ccessions were selected for their colour nd for the cler presence of wxes. Accessions of four Georgin ntive cultivrs, nmely Sirgul B., Rktsiteli Vrdisperi Rs., Ghrubel Krtlis G. nd Khushi Shvi N., were selected. Beside the interest in the vribility of the Georgin grpevine genetic resources, the cultivr selection ws mde ccording to the im of OST Action FA1003, "Est-West ollbortion for Grpevine Diversity Explortion nd Mobiliztion of Adptive Trits for Breeding", within the frmework of the "Development of Phenotyping nd Genotyping Methodologies" working group. Ghrubel Krtlis - the nme of this vriety mens 'foggy from the region of Krtli', feture relted to the opque spect of the berry due to the bundnt wx. Single vines of this vriety re spred mong the old vineyrds of Krtli. The bunch is medium to lrge in size, conicl, seldom cylindricl-conicl, winged, medium dense, nd sometimes loose. The berry is medium or lrge in size, ovte, nd greygreen to violet. The skin is thin, covered with dense wxes, nd esy to peel off. The flesh is juicy nd colourless. Sirgul is vriety from Estern Georgi. Single vines of Sirgul cn be found in the grdens of Kkheti. The bunch is medium in size, cylindricl-conicl or cylindricl, winged or sometimes shouldered, medium dense, but seldom very dense. The berry is medium in size, ovl or seldom rounded, nd light yellow - 'green yellow' ccording to OIV (2007) descriptor code 225. The skin is difficult to peel off. The flesh is firm nd slightly crispy. Rktsiteli Vrdisperi is mutnt Rktsiteli grpe, selected in the mid-twentieth century. The vriety is rrely spred in Kkheti. The bunch is medium in size nd lrge, cylindricl-conicl, rrely cylindricl, winged nd medium dense. The berry is medium, rounded-ovte nd pink. The skin is medium finn. The flesh is colourless, juicy nd with neutrl tste. Khushi Shvi is blck berry vriety from the Western prt of Georgi. It is rre wine grpe vriety from the provinces of Imereti nd Guri. It is minly vilble in field collections now. The bunch is big, cylindricl or cylindriclconic, winged nd medium dense. The berry is medium, rounded or rounded-flttened nd blck. The flesh is slightly rose nd juicy. All the culturl prctices were the sme for the four selected cultivrs. Spring suckering, post-flowering shoot hedging, topping nd positioning, followed by slight prevérison lef removl in the fruiting zone, were the min cnopy mngement steps. Bunch thinning ws not required becuse vine blnce, in terms of crop lod vs. lef re rtio, ws positively evluted by visul inspection. It ws considered higher thn one squre metre of exposed leves per expected kilogrm of grpes nd ble to ssure proper grpe ripening ccording to the locl experience. Ripeness ws evluted in reltion to the vrietl chrcteristics, nd some nlyticl dt re reported in Tble 1. For ech vriety, ten non-deformed nd normlly sized S. Afr. J. Enol. Vitic., Vol. 33, No. 2,2012
Reflectnce ofepicuticulr Wx 140 TABLE 1 Grpe chrcteristion t ripe stge. olour Soluble sugrs ( B) ph Titrtble cidity (gil trtric cid) Rktsiteli Vrdisperi Pink 19.7 3.0 7.8 Ghrubel Krtlis Grey 16.9 3.2 6.9 Khushi Shvi Blck 18.8 2.8 13.3 Sirgul White 19.4 3.2 4.3 berries tken from the middle prt of representtive bunches were selected, nd the reflectnce ws mesured in the sme re before nd fter wx removl. All the res were selected in positions with n evident nd uniform wx lyer. hloroform ws used to remove the epicuticulr wxes (Rosenquist & Morrison, 1988; Brnes et l., 1996; Holmes & Keiller, 2002). Berries were rinsed with pure chloroform nd wiped with rough pper (to obtin n brsive effect) three times to ensure the totl removl of the wxes. The mesurement of the reflectnce ws done with the Jz System (Ocen Optics, B.V.), completed with spectrometer chnnel with DPU module nd 1LX5 1 lb detector, OFLV-3 filter, L2 lens nd 50 I.Lm slit s instlled options. The instrument ws set with n NIRJVis light source intensity of 4095, nd n integrtion time of 100 msec. Ech spectrum reported by the instrument is shown in rbitrry units (.u.), nd is the verge of nine spectr mesured nd medited directly by the instrument. The clculted spectr rnged between 341 nd 1025 nm, with stepwise increse of pproximtely 0.3 nm. The sttisticl signfficnce of the differences between the reflectnce dt from the skins before nd fter wx removl were estimted for ech wvelength ccording to the clssicl computtion of the men confidence intervls per P = 0.95, with the lower limit t M - (t095)(5m) nd the upper limit t M + (t095)(5m) where M = verge vlue, t = Student's t per P = 0.95 nd s m = stndrd error (stndrd devition / number ofbethes 1 ). RESULTS AND DISUSSION The decision to study originl cultivrs from ucsus ws linked to the incresing interest in these vrieties. ucsus represents n importnt centre of genetic vribility for Vitis vinfer (Imzio et l., 2010). For this reson it is importnt to enlrge the reserch studies on vrieties selected in this prt of the world. Georgin cultivrs in fct show n interesting vribility, especilly concerning ripening timing (Mghrdze et l., 2012). Most of the Georgin cultivrs re lte ripening nd the epicuticulr wxes could ply n importnt role in preserving the berry's integrity until the hrvest period. This is further reson to improve existing knowledge of the eco-physiologicl role of berry epicuticulr wxes. For this reson we lso selected four Georgin cultivrs with berries rich in wxes, even if they re not widely cultivted nowdys. Figure 1 shows the reflectnce spectr of some berries before nd fter wx removl. onsidering tht the visul ppernce of the skins ws clerly modified by the presence of the wx, we were expecting the removl of this lyer to cuse n evident chnge in the reflectnce spectrum of the berries. Except for the white berry cultivr, however, the differences relted to the berries seemed to be higher thn the differences relted to the presence of epicuticulr wxes. Hence, the chnge in ppernce cused by this lyer ppers to be relted minly to light-scttering effect, which cn be explined by the chotic orienttion of trnsprent or semi-trnsprent wx pltelets. It my produce n opcifiction of the berries' surfce. However, some chnges in the visible spectr were underlined, s shown in Fig. 2. This figure indictes the verge spectr nd the error for vrietl reflectnce with nd without wxes. A difference in the opticl properties of the wx lyers ws observed mong the four vrieties, in greement with the vribility of the cultivrs' wx structures tht hve lredy been underlined (Rosenquist & Morrison, 1989; Percivl et l., 1993; Mugnu et l., 2011). Rktsiteli Vrdisperi, the pink cultivr, produced lyer tht seemed to increse the reflectnce minly long ll the wvelengths of interest in the reflectnce phenomenon, but the differences were never significnt. In the other three cultivrs, the wx presence significntly incresed the reflectnce in prt of the visible spectrum. The most evident effect ppered in Sirgul, the white vriety. In this cse, the epicuticulr wxes provoked significnt increse in reflectnce between bout 425 nd 655 nm, modifying the opticl properties of the surfce in nerly ll the visible spectr. The increse of reflectnce underlined in Ghrubel Krtlis, the grey cultivr, ws less pronounced. It involved the visible spectrum pproximtely between 475 nd 620 nm. The high reproducibility of the visible spectr in Sirgul, the blck vriety, peimitted the underlining of slight but significnt increse of the reflectnce in the wvelengths included between bout 530 nd 685 nm. In Sirgul nd Rktsiteli Vrdisperi, the increse in reflectnce relted to the wx presence seemed to diminish s the wvelength incresed. One possible explntion cn be relted to the nture of the infrred wves. While the TJV/ visible wves re ble to induce electron trnsition, the less energetic infrred wvelengths ffect vibrtionl trnsitions. Therefore, the opticl behviour of the berry in front of the infrred wves will be different in reltion to the csul sptil orienttion of the molecules. This is in greement with the results shown in Figure 3, which represent the vribility of the opticl effects of the wx, showing the confidence intervls of the difference between the reflectnce spectrum of the berries covered by wxes nd the one obtined fter the chloroform tretments. It clerly ppers tht, in ll the vrieties, the opticl properties of the wxes re much more vrible in the less energetic red nd infrred wvelengths. S. Afr. J. EnoL Vitic., VoL 33, No. 2,2012
141 Reflectnce of Epicuticulr Wx 0) U color: blck, berry: 9 color: gry, berry: 10 color: J\ ZA pink, berry: 9 color: white, berry: 4 U I.- 01 color: blck, berry: 5 10000 8000 6000 4000 2000... I color: gry, berry: 6 color: pink, berry: 8 4 color: 12500 _A 10000-7500- 5000-2500- white. berry: 10 FIGURE 1 Exmple of reflectnce spectr with (grey lines) or without (blck lines) wx in single berry. Except for the white cultivr, the differences relted to the berries seemed to be higher thn the differences relted to the presence of epicuticulr wxes. Thus, the opcifiction of the berries' surfce observed in the berries covered by wxes should be relted minly to lightscttering effect. The ltter is relted to the chotic orienttion of trnsprent or semi-trnsprent wx pltelets. 15000-2D blck color: pink 1 2500 10000-21QD 7500-160D 5000-11DD w.).) 0) w to LI) U) (0 O (0 N N- N- (0 03 00) 0) 0 color: gry color: white 7500-5000- 10000- IIIIIIIIIIIIIII!III FIGURE 2 Men confidence intervls (per P = 0.95) of the reflectnce spectr of the four cultivrs. The grey res indicte the reflectnce mesured in the berries with the wx, while the blck res represent the redings fter wx removl. Only Rktsiteli Vrdisperi (pink) did not show significnt increse in reflectnce, while in the other vrieties the wxes provoked n increse in reflectnce in more-or-less lrge prt of the visible spectr. S. Afr. J. Enol. Vitic., Vol. 33, No. 2,2012
Reflectnce ofepicuticulr Wx 142 color: blck rnlnr nry - 4) U, color: pink color: white, FIGURE 3 Stndrd errors of the difference between the reflectnce spectr before nd fter the chloroform tretments. This represents the vribility of the differences in spectr due to the presence of the wx. The ltter generlly increses in the 1R wvelengths, probbly becuse of the combintion of the nture of these wves nd the chotic orgnistion of the wx pltelets. onsidering tht the totl mount of rdition sent by the instrument to the berry surfce cn only be trnsmitted, reflected or dsorbed, the difference between the light mesured before nd fter wx removl my indicte some properties of the wxes, nmely: - principlly trnsmission when this difference is round 0, becuse the presence of the wx does not chnge the mount of light mesured; - minly reflection when the difference is positive, becuse the presence of the wx increses the reflectnce of the surfce; - some bsorption properties when the difference is negtive, becuse the rdition reflected by the rinsed surfce would hve been stopped by the wxes before rriving bck to the instrument. The high vribility of the opticl properties of the wxes in the ner infrred wvelength region could underline the protective ctivities of this lyer on the bsis of different moleculr nd structurl opticl properties. The described chotic disposition of the wx pltelets on the berry surfce (Mrois et l., 1986; Rosenquist & Morrison, 1988, 1989; Percivl et l., 1993; Rogiers et l., 2004; Mugnu et l., 2011) cn plce molecules in different orienttions regrding the flow of rdition. As fr s infrred wves re concerned, the chotic disposition decreses some vibrtionl trnsitions depending on the chemicl environment, since steric bulk my limit vibrtionl movements. It thus implies high vribility in the medium opticl behviour, limiting the trnsmitted rdition. The structurl rrngement of the wx ws lredy described s wy to control pthogen infections nd wter loss. In the crystlline structures, the cuticulr trnspirtion is shrply reduced through this lyer becuse wter vpour must be diffused through complex network of cpillries creted by the overlpping pltelets. The diffusionl dynmics re reduced, mking this lyer prcticlly impermeble (Hrdie et l., 1996; Rogiers et l., 2004). A side effect of the pltelets' structure nd disposition my hve consequences for the opticl properties. ONLUSION Besides for protecting ginst trnspirtion nd pthogens, the wxes on the grpevine berry proved to hve some opticl properties, with eco-physiologicl side effect. The slight increse in the reflectnce in the visible rnge should S. Afr. J. EnoL Vitic., VoL 33, No. 2,2012
143 Reflectnce of Epicuticulr Wx be tken into ccount, especilly in reltion to the cultivr's vribility. On the other hnd, the overlpping of different opticl behviour in the infrred wvelengths cn be relted to protection ginst berry heting. An improvement in the understnding of the opticl properties of the epicuticulr wxes of the berry seems importnt lso in the definition of the most suitble cultivrs, in prticulr for wrmer viticulturl res. The selection of lte ripening cultivrs, with berries rich in epicuticulr wxes, would llow reduction in the impct of summer stresses during ripening. At the sme time, it would peimit better mngement of n extended ripening phse in the vineyrd by reducing possible berry dehydrtion nd mould decy, with beneficil effect on the oenologicl qulity of the grpes. Moreover, the results encourge studies in this field in reltion to the ctul development of non-invsive instruments to evlute berry ripening nd composition. Especilly visible nd NIR reflectnce hve recently been used s new tool in grpe reserch. Knowledge of the opticl properties of the epicuticulr wxes my help in the development of these methods, where the possible interference of the wxes should be tken into ccount. LITERATURE ITED Brnes, J.D., Percy, K.E., Pul, N.D., Jones, P., McLughlin,.K., Mullineux, P.M., reissen, G. & Wellborn, A.R., 1996. The influence of UV-B rdition on the physicochemicl nture of tobcco (Nicotin tbcum L.) lef surfces. J. Exp. Bot. 47, 99-109. Di Mtteo, M., inqunt, L., Gliero, G. & rescitelli, S., 2000. Effect of novel physicl pretretment process on the drying kinetics of seedless grpes. J. Food Eng. 46, 83-89. Doymz, 1., 2004. Effect of pre-tretments using potssium metbisulphite nd lkline ethyl olete on the drying kinetics of pricots. Biosyst. Eng. 89, 281-287. Doymz, 1., 2006. Drying kinetics of blck grpes treted with different solutions. J. Food Eng. 76, 212-217. Doymz, I. & Pl M., 2002. The effects of dipping pretretments on irdrying rtes of the seedless grpes. J. Food Eng. 52, 413-417. Hrdie, W.J., O'Brien, T.P. & Judzems, V.G., 1996. Morphology, ntomy nd development of the pericq, fter nthesis in grpe, 'itis vin[er L. Aust. J. Grpe Wine Res. 2, 97-142. Holmes, M.G. & Keiller, D.R., 2002. Effect of pubescence nd wxes on the reflectnce of leves in the ultrviolet nd photosynthetic wvebnds: comprison of rnge of species. Plnt ell Environ. 25, 85-93. Imzio, S., Mghrdze, D., Bcilieri, R., De Lorenzis, G., Scienz, A., This P. & Fill 0., 2010. Moleculr survey of Georgin trditionl grpevine genetic resources. Book of bstrcts, the 1 0 0, Interntionl onference on Grpe Genetics nd Breeding, August 2010, Genev. p. 70 Jenks, M.A. & Ashworth, E.N., 1999. Plnt epicuticulr wxes: function, production, nd genetics. Hort. Rev. 23, 1-68. Mghrdze, D., Rustioni, L., Scienz, A. & Fill 0., 2012 Phenologicl diversity of Georgin grpevine cultivrs in northern Itly. J. Amer. Pom. Soc. 66, 56-67. Mhmutolu, T., Emir, F. & Sygi, YB., 1996. Sun/solr drying of differently treted grpes nd storge stbility of dried grpes. J. Food Eng. 29, 289-300. Mrois, J.J., Nelson, J.K., Morrison, J.., Lile, L.S. & Bledsoe, A.M., 1986. The influence of berry contct within grpe clusters on the development of Botrytis cinere nd epicuticulr wx. Am. J. Enol. Vitic. 37, 293-296. Mugnu, M., Bellincontro, A., Brnb, F.E., Polocci, M., Bignmi,., Gmbellini, G. & Mencrelli, F., 2011. Influence of bunch position in the cnopy on berry epicuticulr wx during ripening nd on weight loss during posthrvest dehydrtion. Am. J. Enol. Vitic. 62, 91-98. Muller,. & Reiderer, M., 2005. Plnt surfce properties in chemicl ecology. J. hem. Ecol. 31, 2621-2651. OW, 2007(2nd ed). Descriptor list for grpe vrieties nd Vitis species. The OW, Pris, Frnce. Pngvhne, D.R., Swhney, R.L. & Srsvdi, P.N., 1999. Effect of vrious dipping pretretment on drying kinetics of Thompson seedless grpes. J. Food Eng. 39, 211-216. Percivl, D.., Sullivn, J.A. & Fisher, K.H., 1993. Effect of cluster exposure, berry contct nd cultivr on cuticulr membrne formtion nd occurrence of bunch rot (Botrytis cinere Pers.: Fr.) with 3 Vitis yin ([er L. cultivrs. Vitis 32, 87-97. Rogiers, S.Y., Htfield, J.M., Judzems, V.G., White R.G. & Keller M., 2004. Grpe berry cv. Shirz epicuticulr wx nd trnspirtion during ripening nd prehrvest weight loss. Am. J. Enol. Vitic. 55, 12 1-127. Rosenquist, J.K. & Morrison, J.., 1988. The development of the cuticle nd epicuticulr wx of the grpe berry. Vitis 27, 63-70. Rosenquist, J.K. & Morrison, J.., 1989. Some fctors ffecting cuticle nd wx ccumultion on grpe berries. Am. J. Enol. Vitic. 40, 241-244. Soil Survey Stff, 1999 (2nd ed). Soil txonomy. A bsic system of soil clssifiction for mking nd interpreting soil surveys. USDA - Soil onservtion Service, Agriculturl Hndbook #436, U.S. Government Printing Office, Wshington, D. Strhler, A.H. & Strhler, A., 2003 (3rd ed). Introducing physicl geogrphy. Wiley, New York. S. Afr. J. Enol. Vitic., Vol. 33, No. 2,2012