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Crop Protection 30 (2011) 52e56

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Crop Protection

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Increase in Cladosporium spp. populations and rot of wine grapes associated withleaf removal

B.A. Latorre*, E.X. Briceño, R. TorresFacultad de Agronomía e Ingeniería Forestal, Pontificia Universidad Católica de Chile, Casilla 306-22, Santiago, Chile

a r t i c l e i n f o

Article history:Received 2 March 2010Received in revised form25 August 2010Accepted 27 August 2010

Keywords:CladosporiumDiseasesGrapevinePest managementVitis vinifera

* Corresponding author. Tel.: þ562 585 4159; fax:E-mail address: blatorre@uc.cl (B.A. Latorre).

0261-2194/$ e see front matter � 2010 Elsevier Ltd.doi:10.1016/j.cropro.2010.08.022

a b s t r a c t

Leaf removal reduces the epiphytic populations of several filamentous fungi found on grapevine (Vitisvinifera). Consequently this practice is used to prevent foliar diseases of grapevines and rots of grapes. Inthis study, the effects of leaf removal on Cladosporium rot (Cladosporium cladosporioides and Cladospo-rium herbarum), which often affects ‘Cabernet Sauvignon’ in Chile, were characterized. The effects of leafremoval on epiphytic populations of Cladosporium spp. on grape berry surfaces and on Cladosporium rotdevelopment were investigated. Three leaf removal treatments were compared: (i) severe leaf removal,where leaves from two to three nodes above, opposite and from all nodes below clusters were removed;(ii) mild leaf removal, where leaves opposite each cluster were removed; and (iii) no leaf removal.Regardless of the leaf removal treatment, low population levels of Cladosporium spp. were detected earlyin the ontogenic development of grape berries which increased as the berries matured, reachingmaximum populations on overripe berries. Based on our results, severe leaf removal favors the growth ofCladosporium spp. on grape berries and increases the prevalence of Cladosporium rot at harvest. Thisincrease in Cladosporium spp. was correlated with an increase in lenticel damage in ‘Cabernet Sauvignon’and ‘Sauvignon blanc’ vines subjected to severe leaf removal. Considering that Cladosporium rotsignificantly reduces yield and wine quality, farmers should avoid continuous exposure of grape clustersto sunlight in order to prevent severe outbreaks of Cladosporium rot.

� 2010 Elsevier Ltd. All rights reserved.

1. Introduction

Cladosporium rot, caused by Cladosporium cladosporioides andCladosporium herbarum, is a common disease of grapevines (Vitisvinifera L.) in Chile, particularly in ‘Cabernet Sauvignon’ vines thatare commonly harvested very late in the season, when grapes arepartially senescent (Briceño and Latorre, 2007, 2008). This delay inharvest appears to beneeded toobtain a complete phenolic ripenessof the berries to ensure aroma and flavor development for optimalwine quality (Saint-Cricq et al., 1998). However, a delay in harvestfavors Cladosporium rot, which reduces yield and affects the qualityof wines (Briceño et al., 2009; Pszczólkowski et al., 2001).

Leaf removal has been demonstrated to be an effective canopymanagement strategy to reduce the incidence and severity of foliardiseases and rots of grapevines (Chellemi andMarois,1992; Duncanet al., 1995; Gubler et al., 1987; Stapleton and Grant, 1992; Stapletonet al., 1995). Consequently, farmers normally remove leaves fromthe basal portion of the shoots, leaving clusters exposed to air flowand sunlight after bloom, a practice that affects the microclimate of

þ562 5534130.

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grapevines by increasing temperature and reducing relativehumidity (English et al., 1989).

Numerous fungal genera have been recovered from the surfaceof grape berries, including species of Aspergillus, Botrytis, Clado-sporium, Penicillium, and Alternaria (Díaz et al., 2009; Donoso andLatorre, 2006; Thompson and Latorre, 1999). The populations ofthese species increase as the berry mature. This was demonstratedfor a population of Cladosporium spp. that developed on the surfaceof apparently healthy grape berries (Briceño and Latorre, 2008).

This studywas conducted to determine the effect of leaf removalon the epiphytic populations of Cladosporium spp. and on thedevelopment of Cladosporium rot on wine grape clusters.

2. Material and methods

2.1. Plant material

Leaf removal experiments were conducted in commercial‘Cabernet Sauvignon’ and ‘Sauvignonblanc’ vineyards, in Alto Jahuel(33�4306000S, 70�420000W) and ‘Cabernet Sauvignon’ in Alhué(33�2100000S, 71�0800000W). Both localities are characterized byaMediterranean climate, with rains concentrated inwinter months

Fig. 1. Grapevine (Vitis vinifera) ‘Cabernet Sauvignon’ subjected to a non-defoliated vine treatment (A) and a severe leaf removal treatment (B). Cladosporium rot (C) and lenticeldamage (D) developed on ‘Sauvignon blanc’ grape berries in vines subjected to a severe leaf removal treatment.

B.A. Latorre et al. / Crop Protection 30 (2011) 52e56 53

and warm summer months. The ‘Cabernet Sauvignon’ and ‘Sau-vignonblanc’vineswere 7 and15year-old, respectively, andplantedon their own roots at 2.5�1.2 m, with rows oriented north to southand trained in a bi-lateral cordon trellis systemwith three wires at0.9, 1.2 and 1.5 cm above ground. In all experiments, grapevineswere managed as is customary for wine grapes in Chile (Gil andPszczólkowski, 2007), except that foliar fungicideswerenot applied.

2.2. Leaf removal

The effect of three leaf removal treatments on Cladosporium rotand lenticel damagewere studied: (i) severe leaf removal, consistingof removing leaves from two to three nodes above, opposite eachcluster, and leaves from all nodes below clusters, leaving themexposed to sunlight continuously from fruit set to harvest. (ii) Mildleaf removal, consisting of removing leaves opposite each clusterabout 4e5weeks after fruit set, and (iii) no leaf removal, where non-defoliated plants were left as controls (Fig. 1). In all experiments,

leaves were removed manually on the east and west side of thevines.However, evaluationsweremade separatelyoneachvine side,considering that light interception on the east side of vines orientednorth to south varies between 8 and 10 h, in contrast to 12e19 h onthe west side (Gil and Pszczólkowski, 2007; Smart, 1973).

2.3. Effect of leaf removal on the dynamic of Cladosporium ssp.

The effect of leaf removal on the epiphytic populations of Cla-dosporium spp. was monitored from the date of leaf removal (fruitset) through harvest on grapevines ‘Cabernet Sauvignon’, subjectedto a severe leaf removal treatment in Alto Jahuel in 2007 and 2008and Alhué in 2008. For each sample, 50 berries were randomlyselected, suspended and shaken for 5 min in 50 ml of sterile 0.05%Tween-80 for 5 min. An aliquot (100 mL) of each suspension wassprayed in a 90 mm diameter Petri dish containing acidified potatodextrose agar (2% dehydrated mashed potatoes, 2% glucose and20 g L�1 agar plus 0.5 ml L�192% lactic acid added after autoclaving)

Fig. 2. Effect of a severe leaf removal on the epiphytic populations of Cladosporium spp. on grape berries of V. vinifera ‘Cabernet Sauvignon’. (A and B) Alto Jahuel 2007 and 2008,respectively. (C) Alhué, 2008. Veraison (berry beginning to lose green color) was approximately 63 and 70 days after full bloom (DAFB) in Alto Jahuel in 2007 and 2008, respectively,and 73 DAFB in Alhué in 2008.

B.A. Latorre et al. / Crop Protection 30 (2011) 52e5654

that was supplemented (MPDA) per liter with 0.05 g tetracycline(SigmaeAldrich), and 0.1 g streptomycin (SigmaeAldrich). Onemilliliter of Igepal CO-630 (SigmaeAldrich, Atlanta, USA)was addedas a colony growth restrictor. Plates were incubated at 20 �C for 6days to determine the number of colonies of Cladosporium spp. oneach disk.

2.4. Effect of leaf removal on Cladosporium rot and lenticel damage

At harvest, the prevalence of Cladosporium rot was evaluated in25 berry samples collected arbitrarily from five grape clusters fromeach replicate in each trial. At the same time, the severity of thelenticel damage was evaluated in each berry by counting thenecrotic lenticels under a stereoscopic microscope.

2.5. Experimental design and statistical analysis

Treatments were arranged as randomized complete blocks(blocked within rows) with a 3� 2 (leaf removal treatments� -grapevine sides) factorial arrangement of treatments, with fourreplicates of at least 10 vines each. Data were subjected to a two-way analysis of variance, and means were analyzed separatelyusing Tukey’s multiple comparison test (P< 0.05), using SigmaStat3.1 (Systat Software Inc., San José, CA, USA). If needed, prior toanalysis, data were normalized by log10(xþ 1) transformation;however, non-transformed data are presented.

The effect of leaf removal on the epiphytic populations of Cla-dosporium spp. which developed on ‘Cabernet Sauvignon’ clusterswas described by regression analysis between x¼ days after fullbloom (DAFB) and y¼ Cladosporium spp. populations. Similarly, therelationship between lenticel damage (x) and incidence of

Cladosporium rot (y) obtained in ‘Cabernet Sauvignon’ clusters wasstudied by regression analysis.

3. Results

3.1. Effect of leaf removal on the dynamic of Cladosporium ssp.

Independent of location, year and side of the vine, the pop-ulation dynamics of Cladosporium spp. on the surface of apparentlyhealthy grapes exhibited a similar trend that was characterized bya relatively low density early in the season, increasing rapidly afterveraison (when a berry begins to lose its green color), withmaximum values at harvest. For instance, in grape clusters sub-jected to a severe leaf removal treatment, population densitiesincreased 11- to 34-fold in Alto Jahuel and 132- to 346-fold inAlhué. In contrast, population densities increased 3- to 10-fold and2.6- to 86-fold on berries fromvines that maintained their canopiesunaltered in Alto Jahuel and Alhué, respectively. An exponentialrelationship best described the relation between populationdensities and DAFB (Fig. 2).

The mean population densities of Cladosporium spp. were signif-icantly (P< 0.012) higher on berries exposed to sunlight on thewestside relative to those exposed on the east side of the vines in AltoJahuel. However, the side of the vine had no effect on the populationdensities of Cladosporium spp. obtained in Alhué (Table 1). Leafremoval treatments had a significant (P< 0.001) effect on pop-ulationsofCladosporium spp. inAlto JahuelandAlhué in2008,but theeffect was non-significant in Alto Jahuel in 2007. The interactionbetween leaf removal treatments and side of the vines were signifi-cant in Alto Jahuel (P¼ 0.002) in 2008 and Alhué 2008 (P¼ 0.001)(Table 1).

Table 1Effect of leaf removal on epiphytic populations of Cladosporium spp. on grape(Vitis vinifera) berries ‘Cabernet Sauvignon’.

Treatments Population of Cladosporium spp. on the surface ofapparently healthy berries, cfu cm�2

Alto Jahuel Alhué

2007 2008 2008

Leaf removal levels (LRLs)a

Severe 2758.8 5337.3 6149.2Mild 2503.1 5431.8 3317.1None 1976.7 2202.4 3229.9df 2 3 3F 1.27 10.23 40.37P 0.304 0.001 <0.001SED 1.151 0.349 0.228

Grapevine side (GS)a

East 1588.1a 2985.2 4334.5West 3237.5b 5662.3 4128.3df 1 1 1F 18.80 7.71 2.22P <0.001 0.012 0.154SED 0.329 0.315 0.206

LRL�GS interactiondf 2 2 2F 0.35 8.62 12.59P 0.706 0.002 0.001SED 0.433 0.415 0.037

a Means followed by the same letter in each column did not differ significantlyaccording to Tukey’s test (P¼ 0.05). Data were log10(xþ 1) transformed beforeanalysis, but non-transformed data are presented.

B.A. Latorre et al. / Crop Protection 30 (2011) 52e56 55

Leaf removal treatments increased the population density ofCladosporium spp. by 40e175% in severely defoliated vines relativeto non-defoliated vines. However, differences between populationsfound in berries from unaltered canopies and the populations inberries from severe removal treatments were significant (P¼ 0.05)only for Alto Jahuel and Alhué in 2008.

3.2. Effect of leaf removal on Cladosporium rot

Cladosporium rot appeared on mature grapes (total solublesolids [TSS]> 22%), and was characterized by the presence ofsuperficial olive-green colonies of Cladosporium spp. that confer

Table 2Analysis of variance showing the effect of leaf removal on the development of Cladospor

Treatments Cabernet Sauvignon

Cladosporium rot n� cluster�1 Lenticel damage n� berr

Leaf removal levels (LRLs)a

Severe 2.2 12.0bMild 1.5 9.9aNone 1.4 10.1adf 2 2F 1.28 5.71P 0.301 0.012SED 0.440 4.953

Grapevine side (GS)a

East 0.9 10.5West 2.5 10.8df 1 1F 4.72 0.18P 0.043 0.681SED 0.397 4.475

LRL�GS interactiondf 2 2F 4.03 0.12P 0.035 0.222SED 0.523 5.890

a Means followed by the same letter in each column were not significantly different accbut non-transformed data are presented.

a moldy appearance to the berries that only affected the berriessuperficially (Fig. 1C).

Leaf removal treatments had a significant effect on the incidenceof Cladosporium rot in ‘Sauvignon blanc’ (P< 0.001), but the effectwas not significant on ‘Cabernet Sauvignon’ (P¼ 0.301). However,the grapevine side of the vines significantly (P< 0.043) affected thelevel of Cladosporium rot in both grapevine cultivars. The interac-tion between leaf removal treatment and grapevine side was onlysignificant (P¼ 0.035) in ‘Cabernet Sauvignon’ (Table 2).

Leaf removal significantly increased Cladosporium rot in ‘Sau-vignon blanc’, with a mean of 61.6 diseased berries per cluster;while only four and six diseased berries per cluster were obtainedin vines subjected to mild leaf removal and no leaf removal,respectively (Table 2). In ‘Cabernet Sauvignon’, only mean differ-ences in leaf removal treatments on thewest side of the plants weresignificant (P¼ 0.05).

3.3. Effect of leaf removal on lenticel damage

Lenticel damage was characterized by the development ofnecrotic spherical dots that were brown and about 1.0e2.0 mm indiameter, often surrounded by a reddish halo, which were apparentbefore veraison (Fig.1D). Independent of the grapevine cultivar, leafremoval had a significant (P< 0.012) effect on the severity oflenticel damage, significantly increasing in vines subjected toa severe leaf removal (Table 2). The severity of lenticel damage wasnot affected by the side of the vines where grape cluster werehanging, and the interaction between leaf removal and the side ofthe vines was not significant (Table 2). An exponential model,y¼ 0.0022e0.2303x, R2¼ 0.77, best explained the relation betweenx¼ lenticel damage (x) and y¼ Cladosporium rot (Fig. 3).

4. Discussion

This study demonstrates that a high level of leaf removalincreases epiphytic populations of Cladosporium spp. on the surfaceof wine grape berries and increases the prevalence of Cladosporiumrot at harvest. Based on the negative effect of leaf removal onepiphytic populations of Botrytis cinerea and other grape pathogens(Duncan et al., 1995), mainly caused by increasing air movement

ium rot and lenticel damage of grapes (V. vinifera).

Sauvignon blanc

y�1 Cladosporium rot n� cluster�1 Lenticel damage n� berry�1

61.6b 24.2b4.0a 16.8a6.0a 18.3a2 2

15.59 43.01<0.001 <0.0010.587 5.473

15.1a 19.932.6b 19.61 15.62 0.210.029 0.6490.529 4.946

2 23.82 0.130.894 0.3780.696 2.089

ording to Tukey’s test (P¼ 0.05). Data were log10(xþ 1) transformed before analysis,

e 2 2 0 . 0 = y x 3 0 3 2 . 0

R 2 7 7 . 0 =

0

2

4

6

8

422016218Lenticel damage,no.berry 1-

Cla

dosp

oriu

m ro

t, no

. clu

ster

-1

Fig. 3. Relationship between lenticel damage and Cladosporium rot obtained in grape-vine (V. vinifera) ‘Cabernet Sauvignon’ subjected to a severe leaf removal.

B.A. Latorre et al. / Crop Protection 30 (2011) 52e5656

around grape berries (English et al., 1989), canopy management,including leaf removal, shoot topping and shoot positioning, hasbeen recommended to preventively control Botrytis bunch rot andother grapevine diseases (Chellemi and Marois, 1992; Gubler et al.,1987; Stapleton and Grant, 1992; Stapleton et al., 1995). However,considering that Cladosporium rot significantly reduces yield andwine quality (Briceño et al., 2009; Pszczólkowski et al., 2001),farmers should avoid continuous exposure of grape clusters tosunlight so as to prevent severe Cladosporium rot outbreaks. Theincidence of Cladosporium rot was at least partially associated withan increase in lenticel damage observed in ‘Cabernet Sauvignon’and ‘Sauvignon blanc’ vines subjected to a severe leaf removal. It ispossible that the necrotic lesions that develop around lenticelsmight provide a substrate for the establishment and colonization ofgrape berries by Cladosporium spp. Additionally, Cladosporium spp.produce melanin-like pigments in the cell wall of conidiophoresand conidia, which confer protection against solar radiation(Ulevicius et al., 2000; Valero et al., 2007). Therefore, increasingsolar radiation of the grape clusters through leaf removal may notaffect their survival, which may explain the high densities of Cla-dosporium spp. obtained on grape clusters subjected to severe leafremoval in this study. Regardless of the leaf removal treatment, ourresults confirm the presence of epiphytic populations of Clado-sporium spp. on apparently healthy grape clusters. These pop-ulations were detected very early in the ontogenic development ofgrape berries and increased as the berries matured, reachingmaximum numbers on overripe berries (Briceño and Latorre,2008). As has been suggested for other pathogens affecting grapeberries (Commenil et al., 1997; Jeandet et al., 1991; Mlikota Gableret al., 2003), it is postulated that changes in cuticle porosity, cuticlefractures, lower capacity for developing defense reactions, and thedecrease of epicuticular wax content in ripe to overripe grapes maypromote the presence of sugary and watery exudates that stimulatethe superficial development of Cladosporium spp. in overripeberries. However, further studies are needed to better characterizethe dynamics of the epiphytic populations of Cladosporium ongrape berries.

The populations of Cladosporium spp. were always higher thanthe populations of other filamentous fungi, such as species ofAlternaria, Aspergillus, Botrytis, Penicillium and Rhizopus (results notreported in this article). For instance, population densities of

Cladosporium spp. were 66.1 and 233.6 times higher than Penicil-lium spp. in unaltered ‘Cabernet Sauvignon’, 70 and 152 DAFB inAlto Jahuel, respectively. The environmental conditions, charac-teristic of a Mediterranean climate, with low relative humidity andan absence of summer rains, appear to highly favor the survival anddevelopment of Cladosporium spp. in Chilean vineyards.

In conclusion, leaf removal increases the epiphytic populationsof Cladosporium spp. on grape berry surfaces and it increases Cla-dosporium rot on ‘Cabernet Sauvignon’ and ‘Sauvignon blanc’.Therefore, leaf removal should be used cautiously avoiding overexposure of grape clusters to sun.

Acknowledgments

We thank Viña Santa Rita and Viña Ventisquero for allowing usto use their plantations. This research project was supported byConsorcio Empresarial para la Vid y el Vino, Vinnova project05CTE01-09.

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