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Scientia Horticulturae 125 (2010) 305–308

Contents lists available at ScienceDirect

Scientia Horticulturae

journa l homepage: www.e lsev ier .com/ locate /sc ihor t i

usceptibility of grapevine rootstocks to Cylindrocarpon liriodendri and C.acrodidymum

andra Alaniza,∗, José García-Jiménezb, Paloma Abad-Camposb, Josep Armengolb

Departamento de Protección Vegetal, Facultad de Agronomía, Universidad de la República, Garzón 780, 12900 Montevideo, UruguayInstituto Agroforestal Mediterráneo, Universidad Politécnica de Valencia, Camino de Vera s/n, 46022 Valencia, Spain

r t i c l e i n f o

rticle history:eceived 28 July 2009eceived in revised form 2 March 2010

a b s t r a c t

The susceptibility of the grapevine rootstocks most commonly used in Spain to Cylindrocarpon liriodendriand C. macrodidymum was evaluated. Rooted cuttings of rootstocks 110-R, 1103-P, 140-R, 161-49C, 196-17C, Fercal and SO4 were inoculated by dipping their roots in conidial suspensions (5 × 105 conidia mL−1)

ccepted 6 April 2010

eywords:lack foot diseaseathogenicity

of both pathogens and placed in a greenhouse. One month later, each plant was drench inoculated with20 mL of the corresponding spore solution to guarantee root infection. After four months of incubation,root disease severity index (RSDI) and dry weights of shoot and root were recorded for each plant. RSDIwas the best variable to show the effect of the inoculation with C. liriodendi and C. macrodidymum. Accord-

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itis vinifera ing to this, all rootstocks inthe most susceptible to b

. Introduction

Black foot disease caused by the soilborne fungi Cylindrocar-on liriodendri J.D. MacDonald & E.E. Butler (Halleen et al., 2006b)nd C. macrodidymum Schroers, Halleen & Crous (Halleen et al.,004) has become one of the most important trunk diseases thatffects grapevines in nurseries and young vineyards worldwideRego et al., 2000; Petit and Gubler, 2005; Halleen et al., 2006a;laniz et al., 2007). Recently, a third Cylindrocarpon species has beenescribed associated to grapevine roots, C. pauciseptatum SchroersCrous (Schroers et al., 2008). The pathogenicity of this species to

rapevine rootstock 110-R was confirmed by Alaniz et al. (2009a),ut the role of C. pauciseptatum as a causal agent of black footisease has not been elucidated so far.

Cylindrocarpon spp. infect the grapevine plants through the rootnd the basal ends of grapevine rootstocks, causing sunken necroticoot lesions and a reduction of root biomass (Halleen et al., 2003).emoval of rootstock bark reveals black discoloration and necro-is of wood tissues, which develop from the base of the rootstock.ther symptoms include reduced vigour, shortened internodes,

parse foliage and small leaves with interveinal chlorosis andecrosis, frequently leading to the death of the plants (Grasso, 1984;

rasso and Magnano Di San Lio, 1975; Halleen et al., 2006a; Malutand Larignon, 1991; Rego et al., 2000; Scheck et al., 1998).

The susceptibility of grapevine rootstocks and varieties toeveral important grapevine trunk and root pathogens such as

∗ Corresponding author. Tel.: +598 23551108.E-mail addresses: salaniz@fagro.edu.uy, sandra1971@hotmail.com (S. Alaniz).

304-4238/$ – see front matter © 2010 Elsevier B.V. All rights reserved.oi:10.1016/j.scienta.2010.04.009

ated were affected by the disease in some degree, being the rootstock 110-Rathogens.

© 2010 Elsevier B.V. All rights reserved.

Phaeomoniella chlamydospora, Phaeoacremonium spp., Eutypa lataand Armillaria mellea has been evaluated, demonstrating the exis-tence of different levels of susceptibility among the differentmaterials studied (Eskalen et al., 2001; Feliciano et al., 2004;Baumgartner and Rizzo, 2006; Sosnowski et al., 2007). Regardingblack foot disease, Gubler et al. (2004) indicated that the rootstocksVitis riparia “039-16” and “Fredon” have a good degree of resistanceto this disease. Moreover, Jaspers et al. (2007) evaluated the sus-ceptibility of the more commonly planted grapevine rootstocks inNew Zealand in greenhouse conditions. The results showed thatall rootstock varieties included in the study were susceptible toCylindrocarpon spp. in some degree.

At the present time, there are no effective control measuresavailable against black foot disease. The use of plant material withfield resistance can contribute to its control. Thus, the objective ofthe present study was to evaluate the susceptibility of the grapevinerootstocks most commonly used in Spain to C. liriodendri and C.macrodidymum under greenhouse conditions.

2. Material and methods

2.1. Plant material and fungal isolates

Seven grapevine rootstocks: 110 Richter (110-R), 1103 Paulsen

(1103-P), 140 Ruggeri (140-R), 161-49 Couderc (161-49C), 196-17Castel (196-17C), Fercal and SO4, commonly used for grapevinecultivation in Spain were evaluated. The plant materials consistedin eight-month-old dormant rooted cuttings that were supplied bya commercial nursery.

306 S. Alaniz et al. / Scientia Horticulturae 125 (2010) 305–308

Table 1GENMOD procedure analysis and analyses of variance for the effects of experiment, inoculation with Cylindrocarpon liriodendri (Cy 34) or C. macrodidymum (Cy 101) androotstock, on root disease severity index, and root and shoot dry weights, respectively.

Root disease severity indexa Root dry weightb Shoot dry weightb

dfc P-valued df MSe P-valuef df MS P-valuef

Cylindrocarpon liriodendriExperiment (A) 1 0.9470 1 0.1244 0.8947 1 9.0384 0.1930Inoculation (B) 1 <0.001 1 101.073 <0.001 1 47.966 0.0030Rootstock (C) 6 <0.001 6 166.078 <0.001 6 132.537 <0.001A × B 1 0.3647 1 3.2302 0.5004 1 0.2484 0.8287A × C 6 0.5579 6 2.4665 0.9102 6 2.6720 0.8040B × C 6 0.4800 6 22.7556 0.0052 6 11.4424 0.0491Residual 170 7.0836 164 5.2909

Cylindrocarpon macrodidymumExperiment (A) 1 0.5234 1 0.44283 0.7979 1 15.5428 0.0611Inoculation (B) 1 <0.001 1 28.3103 0.0385 1 26.4754 0.0149Rootstock (C) 6 <0.001 6 224.572 <0.001 6 147.973 <0.001A × B 1 0.9282 1 0.9426 0.7040 1 0.1102 0.8740A × C 6 0.6470 6 4.1701 0.6976 6 3.5103 0.5690B × C 6 0.4934 6 5.3840 0.5504 6 8.1376 0.0902Residual 167 6.5094 170 4.3723

a Root disease severity index data were analyzed with the GENMOD procedure using the multinomial distribution and the cumulative logit as link function.b Root and shoot dry weight data were analyzed with analyses of variance (ANOVA).

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c Degrees-of-freedom.d Probabilities associated with individual F-tests.e Mean square.f Probabilities associated with individual Chi-square tests.

For rootstock inoculation, one isolate of C. liriodendri (Cy 36) andne of C. macrodidymum (Cy 101) were used. These isolates werebtained from the Cylindrocarpon spp. collection of the Institutogroforestal Mediterráneo, Universidad Politécnica de Valencia,pain, and were selected among the most virulent isolates accord-ng to previous studies conducted by Alaniz et al. (2009b). They

ere stored in 15% glycerol solution at −80 ◦C into 1.5 mL cryovials.

.2. Pathogenicity tests

Fungal isolates were grown on PDA for one month at 25 ◦C prioro inoculation. The inoculum was prepared for each isolate by flood-ng the agar surface with 10 mL of sterile distilled water (SDW) andcraping with a spatula. The resulting spore suspension was fil-ered through two layers of cheese cloth. The filtrate was dilutedith sterile distilled water (SDW) and conidial concentration was

djusted with a hemacytometer to 5 × 105 conidia mL−1.Before inoculation, roots of rootstock cuttings were trimmed

nd disinfested by immersion during 2 min into a 1.5% sodiumypochlorite solution, and washed twice with SDW. Eight plantsf each rootstock per Cylindrocarpon species were inoculated byipping their roots for 30 min in the conidial suspension. Controllants were dipped in SDW. Then, the inoculated rooted cuttingsere planted individually in 15 cm diameter plastic pots contain-

ng sterilized peat moss and placed in a greenhouse at 25–30 ◦C incompletely randomized design. One month later, each plant wasrench inoculated with 20 mL of the corresponding spore solution5 × 105 conidia mL−1) to guarantee root infection, or SDW (Alanizt al., 2007).

After four months of incubation, all plants were gently uprootednd washed free of substrate. Root disease severity index (RDSI) ofndividual plants was evaluated on the following scale: 0 = healthy

ith no lesions, 1 = slight discoloration with 0–10% of root masseduction, 2 = slight discoloration with 10–25% of root masseduction, 3 = moderate discoloration with 25–50% of root mass

eduction, 4 = severe discoloration with >50% of root mass reduc-ion, 5 = dead plant. In addition, dry weights of shoot and root wereecorded for each plant. Symptomatic roots were aseptically platedn MEAS to reisolate the inoculated fungus. Pathogenicity testsere conducted twice.

Root disease severity data were analyzed with the GENMOD pro-cedure using the multinomial distribution and the cumulative logitas link function in SAS (SAS Institute Inc., Cary, NC, USA). Analy-ses of variance (ANOVA) were conducted on root and shoot dryweights using the Statgraphics Plus 5.1 software (Manugistics Inc.,Rockville, MD, USA). Lineal single-degree-of-freedom contrastswere computed in each rootstock to test the effect of inoculationwith C. liriodendri or C. macrodidymum (Mead et al., 2003). In allanalyses the outlier data were eliminated.

3. Results

For each Cylindrocarpon species inoculated, GENMOD andANOVA analyses indicated that the data between experimentswere similar (P > 0.05) (Table 1), thus data of all variables fromboth experiments were combined. GENMOD and ANOVA analy-ses also indicated significant effects of inoculation and rootstockin all variables evaluated (P < 0.05). Moreover, significant inocula-tion × rootstock interaction was observed for shoot and root dryweight variables in the rootstocks inoculated with the species C.liriodendri (P < 0.05) (Table 1).

Characteristic foliar and root symptoms of black foot diseasewere observed in rootstock plants inoculated with C. liriodendri or C.macrodidymum. The symptoms included reduced vigour of foliage,reduced root biomass and necrotic leaves and roots.

The effect of rootstock inoculation with C. liriodendri or C. macro-didymum on RDSI and shoot and root dry weights is shown in Fig. 1.Inoculation with C. liriodendri or C. macrodidymum resulted in anincrease of RDSI values compared with non-inoculated cuttings inall seven grapevine rootstocks, which were significantly differentin most of the cases. For both Cylindrocarpon species, rootstock 110-R was the most susceptible to inoculation, showing the highestRSDI values: 1.0 and 0.93 for C. liriodendri and C. macrodidymum,respectively.

In general, for all rootstocks the root dry weight values were

lower in the cuttings inoculated with C. liriodendri or C. macro-didymum than in non-inoculated, but significantly different onlyfor the 110-R rootstock inoculated with both pathogens and Fer-cal rootstock inoculated with C. liriodendri. In the case of shoot dryweight, in most of the cases the values were lower in cuttings inocu-

S. Alaniz et al. / Scientia Horticulturae 125 (2010) 305–308 307

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ig. 1. Root disease severity index (RDSI), and root and shoot dry weights of sevenacrodidymum (CM) isolate Cy 101. Results are the means of sixteen rootstock pl

ingle-degree-of-freedom contrasts were computed to test in each rootstock the ef

ated with C. liriodendri or C. macrodidymum than in non-inoculated,ut significantly different only in the 1103-P grapevine rootstock

noculated with both pathogens and Fercal rootstock inoculatedith C. liriodendri. Both Cylindrocarpon spp. were reisolated from

noculated plants on MEAS confirming Koch’s postulates.

. Discussion

The susceptibility of the grapevine rootstocks most commonlysed in Spain to C. liriodendri and C. macrodidymum, was evalu-ted under greenhouse conditions. Both pathogens induced typicallack foot disease symptoms in all grapevine rootstocks studied,owever, the response to inoculation varied among grapevine root-tocks and depending on the variable analyzed.

According to the RDSI data, which include reduction of rootass and other symptoms such as discoloration, necrosis and gen-

ral appearance of roots, most of grapevine rootstocks evaluatedere significantly affected by C. liriodendri and C. macrodidy-um inoculation, being rootstock 110-R the most susceptible.

hese results are in agreement with those obtained by Jasperst al. (2007), who demonstrated that the more common root-tocks planted in New Zealand were susceptible to Cylindrocarpon

pp. in some grade when inoculated under greenhouse condi-ions.

In general, the results obtained for root or shoot dry weightsere variable among the different rootstocks and significant only

or some of them. The root dry weight of the 110-R rootstock

evine rootstocks inoculated with Cylindrocarpon liriodendri (CL) isolate Cy 36 or C.eight per experiment). Vertical bars are the standard error of the means. a Linealinoculation with C. liriodendri or C. macrodidymum, * significant at P < 0.05.

was significantly affected by both Cylindrocarpon species; how-ever, in the case of shoot dry weight only the rootstock 1103-Pwas significantly affected by both Cylindrocarpon species. Fercalrootstock, which was affected only by C. liriodendri, showed asignificant reduction in both root and shoot dry weights. As a soil-borne pathogen, Cylindrocarpon spp. infect the roots and the basalends of grapevine rootstocks (Halleen et al., 2003), generating thesymptoms described before in roots and aerial parts of the plants.However, previous grapevine pathogenicity tests conducted withCylindrocarpon spp. under greenhouse conditions, indicated that,in general, there is not good correlation between root mass reduc-tion and root necrosis in root, and shoot and foliar symptoms (Petitand Gubler, 2005; Alaniz et al., 2007), as also found in our work.As a result, the RSDI variable was the best to show the effect of theinoculation with both Cylindrocarpon species.

The results obtained in this work showed that all rootstocksinoculated with C. liriodendi and C. macrodidymum were affectedby the disease in some degree, being the rootstock 110-R the mostsusceptible to both pathogens. Rootstock 110-R is currently one ofthe more commonly used in Spain because of its good affinity withV. vinifera and high tolerance to drought (Hidalgo, 2002; Jackson,2008), but it seems to be very susceptible to C. liriodendri and C.

macrodidymum. Moreover, this rootstock has been also describedas highly susceptible to Petri disease pathogens such as Phaeoacre-monium aleophilum (Eskalen et al., 2001) and P. chlamydospora(Zanzotto et al., 2008). Consequently, this information should beconsidered when selecting rootstocks for new plantations.

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cknowledgements

Financial support is acknowledged from the Projects AGL2006-1884-C04-01 (Ministerio de Educación y Ciencia, Spain) andTA2007-00023-C04-03 (Programa Nacional de Recursos y Tec-ologías Agrarias, Ministerio de Educación y Ciencia, Spain). Sandralaniz was supported by a grant from the Agencia Espanola deooperación Internacional (AECI—Ministerio de Asuntos ExterioresCooperación, Spain). We acknowledge E. Albelda for technical

ssistance and Dr. R. Raposo for providing valuable advice on theriting of this manuscript.

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