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Crop Protection 30 (2011) 489e494

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Crop Protection

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Evaluation of fungicides to control Cylindrocarpon liriodendri and Cylindrocarponmacrodidymum in vitro, and their effect during the rooting phase in the grapevinepropagation process

Sandra Alaniz a, Paloma Abad-Campos b, José García-Jiménez b, Josep Armengol b,*aDepartamento de Protección Vegetal, Facultad de Agronomía, Universidad de la República, Garzón 780, 12900-Montevideo, Uruguayb Instituto Agroforestal Mediterráneo, Universidad Politécnica de Valencia, Camino de Vera s/n, 46022-Valencia, Spain

a r t i c l e i n f o

Article history:Received 13 August 2010Received in revised form8 December 2010Accepted 19 December 2010

Keywords:Black footGrapevine nurseriesPetri diseaseVitis vinifera

* Corresponding author.E-mail address: jarmengo@eaf.upv.es (J. Armengol

0261-2194/$ e see front matter � 2010 Elsevier Ltd.doi:10.1016/j.cropro.2010.12.020

a b s t r a c t

The efficacy of 14 selected fungicides against Cylindrocarpon liriodendri and Cylindrocarpon macro-didymum was evaluated in vitro by testing their effect on mycelial growth and conidial germination.Carbendazim, hydroxyquinoline sulphate, imazalil, and prochloraz were the most effective fungicides inreducing mycelial growth in both Cylindrocarpon species. Captan, copper oxicloride, didecyldimethy-lammonium chloride and thiram were the most effective to inhibit conidial germination of both species.A pot assay was also conducted with captan, carbendazim, copper oxychloride, didecyldimethylammo-nium chloride, hydroxyquinoline sulphate, imazalil and prochloraz in order to determine their potentialto prevent infections caused by C. liriodendri and C. macrodidymum during the rooting phase in thegrapevine propagation process. All fungicides significantly decreased the root disease severity values inboth species compared with control treatment, with the exception of imazalil in C. macrodidymum. In thecase of the percentage of reisolation, all values were lower than those obtained for the control treatment,but only captan, carbendazim and didecyldimethylammonium chloride were significantly different in thecase of the cuttings inoculated with C. liriodendri, and prochloraz in the case of those inoculated withC. macrodidymum.

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1. Introduction

Over the last 15 years, an important decline of grapevines innurseries and young vineyards has been noticed worldwide.According to Waite and Morton (2007), the causes are numerousand complex, including poor nursery sanitation, inappropriateplanting practices and pathogen infections.

Cylindrocarpon liriodendri J.D. MacDonald & E.E. Butler andCylindrocarponmacrodidymum Schroers, Halleen& Crous, the causalagents of black foot disease of grapevines, havebeen reported as twoof the main fungal pathogens involved in nursery and young vine-yards (Halleen et al., 2006). Both pathogens have been consistentlyisolated from the roots and the basal ends of grafted cuttings innurseries and young vineyards (Rego et al., 2000; Halleen et al.,2004; Petit and Gubler, 2005; Aroca et al., 2006; Alaniz et al.,2007). Infected plants show sunken necrotic root lesions witha reduction in root biomass and root hairs. Removal of rootstock barkreveals black discoloration and necrosis of wood tissues which

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develop from the base of the rootstock. Other symptoms includereduced vigor, shortened internodes, sparse foliage and small leaveswith interveinal chlorosis and necrosis, frequently leading to thedeath of the plants (Grasso,1984;Maluta and Larignon,1991; Schecket al., 1998; Rego et al., 2000; Halleen et al., 2006).

The large number of wounds made in the propagation materialduring the different nursery stages make it very susceptible to beinfected by several fungi, including Cylindrocarpon spp. Species inthis genus have been isolated from grapevine propagation materialin different stages of the nursery propagation process (Halleenet al., 2003; Giménez-Jaime et al., 2006; Aroca et al., 2006) andfrom grapevine rootstock mother plants (Fourie and Halleen,2004a). In grapevine nurseries, the rooting phase is apparentlyone of the most susceptible stages because the fragility of the calluswhich frequently breaks during the planting process resulting insmall wounds susceptible to infection by Cylindrocarpon spp.(Halleen et al., 2003).

During the last years, research has been specially focused in thedevelopment of procedures and chemical products to prevent orreduce black foot disease infection of grapevine woody tissuesduring thepropagationprocesswithpromising results including, the

S. Alaniz et al. / Crop Protection 30 (2011) 489e494490

use of hot-water treatments (Halleen et al., 2007; Bleach et al., 2009;Gramaje et al., 2010), biological control (Halleen et al., 2007),applications of chitosan (Nascimento et al., 2007), use of arbuscular-mycorrhizal fungi (Petit and Gubler, 2006) or fungicides (Fourie andHalleen, 2006; Halleen et al., 2007; Rego et al., 2006, 2009).

Regarding the fungicides, Rego et al. (2006) found that thefungicides benomyl, tebuconazole and themixtures of carbendazimwith flusilazole and cyprodinil with fludioxonil, selected accordingto in vitro studies, significantly decreased black foot incidence ongrapevine plants when roots were treated with these fungicidesbefore being planted in inoculated peat pots. In a later study, theseauthors found that fludioxonil and the mixtures of cyprodinil withfludioxonil and pyraclostrobin with metiram reduced the incidenceand severity of Cylindrocarpon spp. on grapevine plants grown ina commercial field with grapevine cultivation history (Rego et al.,2009). Halleen et al. (2007) performed an in vitro evaluation of 13fungicides against C. liriodendri and C. macrodidymum. Benomyl,flusilazole, imazalil and prochloraz were effective in reducingmycelial growthof black foot pathogens.Nevertheless, onlybenomyland imazalil showed some effect to control these pathogens in semi-commercial field trials.

Presently, there are no effective measures to control black footpathogens in grapevine nurseries or young vineyards. Thus, the aimof this studywas toevaluate the invitroefficacyof fourteen fungicidesagainst C. liriodendri and C. macrodidymum. Additionally, a pot assaywas conducted with seven selected fungicides in order to determinetheir potential to prevent infections caused by these pathogensduring the rooting phase in the grapevine propagation process.

2. Materials and methods

2.1. In vitro evaluation of fungicides

2.1.1. IsolatesIn this study, two isolates of C. liriodendri (Cy 59 and Cy 88) and

two of C. macrodidymum (Cy 14 and Cy 63) were used. Isolates wereobtained from different geographic origins and grapevine root-stocks showing black foot symptoms in Spain (Alaniz et al., 2007).These isolates were single-spored prior to be used by means of theserial dilution method (Dhingra and Sinclair, 1995) and stored in15% glycerol solution at �80 �C in 1.5 ml cryovials.

2.1.2. FungicidesCommercial formulations of 14 fungicides representing nine

different chemical classes were evaluated to test in vitro theircapability to inhibit both mycelial growth and conidial germinationof C. liriodendri and C. macrodidymum (Table 1).

2.1.3. Mycelial growth assayAppropriate volumes of each fungicide were added to molten

potato dextrose agar (PDA) at approximately 50 �C in order toobtain a final concentration of 100, 10, 1 and 0.1 mg of activeingredient per liter (a.i. l�1). Mycelial plugs (4 mm in diameter),obtained from the margins of 10-day old actively growing cultures,were transferred to fungicide-amended plates. Control PDA plateswere prepared similarly but adding sterile distilled water (SDW)instead of the fungicide solution. There were two replicates of eachfungicide concentration, and the experiment was repeated twice.The dishes were incubated for 10 days at 25 �C in the dark, and thediameter of each colony was measured twice perpendicularly.Measurements were made at the same time and averaged.

2.1.4. Conidial germination assayFungal isolates were grown on PDA and incubated for 10 days at

25 �C in the dark. A conidial suspension was prepared for each

isolate by flooding the agar surface with 10 ml of SDWand scrapingwith a sterile spatula. The resulting spore suspension was filteredthrough two layers of cheese cloth into a 250 ml Erlenmeyer flask.The filtrate was diluted with SDW and conidial concentrationwas adjusted with a hemacytometer to 5�105 conidiaml�1. Eachconidial suspension (50 ml) was dispersed into 250 ml Erlenmeyerflasks and appropriate volumes of each fungicide were added toachieve the final concentrations mentioned above, or withoutfungicide as control. Two 40 ml droplets of these suspensions weresubsequently placed in the base of slides and incubated in a moistchamber at 25 �C in the dark. There were two replicates (Erlen-meyer flasks) of each fungicide concentration, and the experimentwas repeated twice. Conidial germination was assessed after 24 hfor each combination of fungicide concentration and isolate. Twohundred conidia were assessed for germination in each droplet.A conidium was considered germinated when the germ tube hadexceeded one-half the length of the spore.

2.1.5. Data analysesPercentage inhibition of mycelial growth and conidial germi-

nation for each isolate at each concentration was calculated asa percentage with respect to the control treatment. Percentages ofmycelial growth and conidial germination inhibitions were con-verted to probits and plotted against log10 values of the fungicideconcentration. Probit regression analysis was used to calculate theeffective concentration values that inhibited mycelial growth andconidial germination by 50% (EC50 values).

EC50 values were analyzed by analysis of variance (ANOVA)performed with the General Linear Model (GLM) by SAS (StatisticalAnalysis System, version 9.0, 14 SAS Institute Inc., Cary, NC, USA).Factors considered in the model were: experiment (performedtwice), fungicide (described in Table 1), pathogen (C. liriodendri andC. macrodidymum) and isolate nested in pathogen (2 isolates foreach pathogen). F tests for each term of the model were derivedfrom the expectedmean squares obtained by application of the rulefor finding expected mean squares. Means were compared usingthe least significant difference (LSD) value at P¼ 0.05.

2.2. Effect of selected fungicides on the rooting phase

According to the results obtained from the in vitro experiments,seven fungicides were selected to determine their potential toprevent infections caused by C. liriodendri and C. macrodidymum ongrapevine cuttings during the rooting phase. Suspensions of thefungicides captan (2 g a.i. l�1), carbendazim (0.3 ml a.i. l�1), copperoxychloride (4 g a.i. l�1), didecyldimethylammonium chloride(0.18 ml a.i. l�1), hydroxyquinoline sulphate (0.5 ml a.i. l�1), imazalil(0.45 ml a.i. l�1) and prochloraz (0.23 g a.i. l�1) were preparedaccording to label rates for other grapevine diseases or similarfungi.

Cuttings of 110 Richter rootstock were hot-water treated at53 �C for 30 min in a hot water bath (PSELECTA Unitronic 320OR,standard error� 0.1 �C, Barcelona, Spain) in order to eliminateany existing infections by fungal trunk pathogens (Gramaje et al.,2009a). Hot-water treated cuttings were stored 24 h at 20 �Cbefore use for acclimatization and then, their basal ends weredipped for 1 h in different fungicide suspensions. Cuttings dippedin SDW were used as a control.

The isolates Cy 36 of C. liriodendri and Cy 96 of C. macrodidymum(Alaniz et al., 2007) were grown on PDA for 10 days at 25 �C in thedark. Conidial suspensions were prepared as described before andwere added to sterilized peat to obtain a concentration of 5�105

conidia g�1 of substrate for each Cylindrocarpon species. The inoc-ulated substrate was placed in 15 cm diameter plastic pots. Onetreated cutting was planted in each pot and was immediately

Table 1Fungicides selected for in vitro sensitivity testing.

Chemical Group Active ingredient Trade name Manufacturer Formulationa Registered concentrationin Spainb

Benzimidazole Carbendazim Quimuzin Sarabia (Lleida, Spain) 500 g l�1 SC 0.6 ml l�1 (cereals)Thiophanate-methyl Pelt Bayer (Valencia, Spain) 450 g kg�1WG 1e1.5 ml l�1 (grapes)

Copper Copper oxychloride Curenox 50 Ind. Químicas del Vallés(Barcelona, Spain)

500 g kg�1 SL 3e4 g l�1 (grapes)

Cubiet Talo-Sint Morera (Valencia, Spain) 500 g l�1 SL 15 l ha�1 (grapes)Cyclic imides Captan Captan 50 Ind. Químicas del Vallés

(Barcelona, Spain)500 g kg�1WP 3e4 ml l�1 (grapes)

Dicarboximide Iprodione Parmex DowAgroSciences(Madrid, Spain)

500 g kg�1 SC 1e1.5 g l�1 (grapes)

Dimetihylthiocarbamatec Thiram Pomarsol F Bayer (Valencia, Spain) 800 g kg�1WP 2e3 g l�1 (grapes)

DMId

Imidazole Prochloraz Sporgon Agrevo (Valencia, Spain) 460 g kg�1WP 0.75e1.25 kg ha�1 (rice)Triazole Flusilazole Olymp Du Pont (Barcelona, Spain) 10 g kg�1 SL 0.15e0.5 ml l�1 (grapes)

Imazalil Fecundal S Janssen-Cilag (Beerse, Belgium) 75 g l�1 SC 5e6 ml l�1 (fruits)Tebuconazole Folicur Bayer (Valencia, Spain) 250 g kg�1WP 0.4e1 g l�1 (grapes)

Strobilurin Azoxystrobin Ortiva Syngenta (Madrid, Spain) 250 g kg�1 SC 0.75e1 g l�1 (grapes)Quaternary ammonium Didecyldimethylammonium

chlorideSporekill ICA International Chemicals

(Stellenbosh, South Africa)120 g l�1 EW Not registered

Quinolitic Hydroxyquinoline sulphate Beltanol L Probelte (Murcia, Spain) 500 g kg�1 SL 6e12 ml l�1 (grapes)

a WP, wettable powder; WG, water dispersible granule; EC, emulsifiable concentrate; SC, suspension concentrate; EW, emulsion oil in water; and SL, soluble concentrate.b Registered concentrations in Spain were determined by De Liñan (2008).c Also listed as a disulphide.d Demethylation inhibitors.

Table 2Analysis of variance for the effects of experiment, fungicide, pathogen and isolate(pathogen) on mycelial growth and conidial germination of Cylindrocarpon lir-iodendri and C. macrodidymum.

dfa EC50

MSb P< Fc

Mycelial growthExperiment 1 40.89 0.4450Fungicide 13 22834.87 <0.001Pathogen 1 15209.39 <0.001Isolate (pathogen) 6 247.22 0.0033Experiment� fungicide 13 48.78 0.7566Experiment� pathogen 1 34.15 0.4851Experiment� isolate (pathogen) 6 86.03 0.2943Fungicide� pathogen 13 8240.64 <0.001Fungicide� isolate (pathogen) 78 111.75 0.0146

Residual 91 69.48

Conidial germinationExperiment 1 12.38 0.7911Fungicide 13 33417.99 <0.001Pathogen 1 1034.19 0.0172Isolate (pathogen) 6 622.01 0.0034Experiment� fungicide 13 193.28 0.3683Experiment� pathogen 1 11.75 0.7964Experiment� isolate (pathogen) 6 232.60 0.2540Fungicide� pathogen 13 679.46 <0.001Fungicide� isolate (pathogen) 78 570.64 <0.001

Residual 91 175.51

a Degrees of freedom.b Mean square.c Probabilities associated with individual F tests.

S. Alaniz et al. / Crop Protection 30 (2011) 489e494 491

irrigated with 20 ml of the corresponding fungicide suspensions.Controls were planted in inoculated peat and irrigated only withSDW. Ten cuttings were used per Cylindrocarpon species andfungicide. All pots were covered with plastic bags to maintain highhumidity, and incubated at 25 �C in a temperature controlledgrowth chamber simulating the conditions for the rooting phase ingrapevine nurseries. The experiment was repeated.

After one month of incubation, the effect of fungicides wasevaluated recording a root disease severity index (RDSI) of indi-vidual cuttings with a scale from 0 to 5 (0¼ healthy with no lesions,1¼ slight discoloration with 0e25% of root mass reduction,2¼ slight to moderate discoloration with 26e50% of root massreduction, 3¼moderate discoloration with 51e75% of root massreduction, 4¼ severe discoloration with >75% of root mass reduc-tion and 5¼ dead plant). In addition, the percentage of reisolationof C. liriodendri or C. macrodidymum from roots of individualcuttings (seven isolation points on PDA supplemented with0.5 g l�1 of streptomycin sulphate per cutting) was calculated.

2.2.1. Data analysesThe RDSI data were analyzed with GENMOD procedure using

the multinomial distribution and the cumulative logit as linkfunction, andmeans of the values were separated by chi-square testat P< 0.05. Analyses of variance (ANOVA) were conducted toanalyze the percentage of reisolation of C. liriodendri or C. macro-didymum andmeans of the values were separated by Student’s leastsignificant difference test at P< 0.05. Percentage data were trans-formed into arcsine (Y/100)1/2 before analysis. In all cases the SASprogram (SAS Institute Inc., Cary, NC, USA) was used.

3. Results

3.1. In vitro evaluation of fungicides

3.1.1. Mycelial growth assayAnalysis of variance showed that there were no differences in

the inhibition of mycelial growth between the two conductedexperiments, but the effect of fungicides, pathogen and theirinteraction were all highly significant (P< 0.001). There were alsosignificant differences between pathogen isolates and the effect of

the interaction between fungicide and pathogen isolates (P< 0.05)(Table 2). Mean EC50 values for reduction in mycelial growth ofC. liriodendri and C. macrodidymum are given in Table 3. Carben-dazim, didecyldimethylammonium chloride, hydroxyquinolinesulphate, imazalil and prochloraz were the most effective fungi-cides in inhibiting mycelial growth of C. liriodendri isolates pre-senting EC50 values between 0.14 and 2.29 mg a.i. l�1. All the otherfungicides were significantly less effective in inhibiting mycelialgrowth, with EC50 values ranging from 26.50 to >100 mg a.i. l�1.Azoxystrobin, carbendazim, flusilazole, hydroxyquinoline sulphate,

Table 3EC50 values for inhibiting in vitro mycelial growth and conidial germination of Cylindrocarpon liriodendri and C. macrodidymum by fungicides representing different chemicalclasses.

Fungicide Mycelial growtha LSD(1)c Conidial germinationa LSD(2)d

C. liriodendri C. macrodidymum C. liriodendri C. macrodidymum

Cy 59 Cy 88 Cy 14 Cy 63 Cy 59 Cy 88 Cy 14 Cy 63

Azoxystrobin 76.00 B bb >100 A a 1.50 C d 2.73 C de 21.39 35.15 B c 79.50 AB a 59.75 AB b >100 A a 49.36Captan 100 A a 86.75 A b 90.75 A ab 100 A a 16.49 1.65 A d 1.50 A b 0.46 B c 0.49 B c 0.51Carbendazim 0.51 B d 0.42 B e 0.55 B d 1.19 A e 0.49 77.50 A b 84.25 A a >100 A a >100 A a 37.55Copper oxychloride >100 a >100 a >100 a >100 a nd 17.50 A cd 2.92 B b 0.90 B c 2.06 B bc 12.50Cubiet >100 a >100 a >100 a >100 a nd 37.25 A c 6.65 B b 4.45 B c 2.56 B bc 10.75Didecyldimethylammonium

chloride1.86 B d 2.10 B e 87.00 A b 100 A a 13.48 1.03 A d 0.62 AB b 0.78 AB c 0.41 B c 0.45

Flusilazole 34.5 A c 30.20 A d 0.25 B d 0.41 B e 11.07 >100 a >100 a >100 a >100 a ndHydroxyquinoline sulphate 0.29 B d 2.29 A e 1.73 A d 0.49 B e 0.81 >100 a >100 a >100 a >100 a ndImazalil 1.30 A d 1.01 A e 1.30 A d 1.08 A e 0.85 10.13 B d >100 A a 10.70 B c >100 A a 6.98Iprodione >100 A a >100 A a 9.50 C cd 18.50 B c 6.02 95.00 A ab 81.00 A a >100 A a >100 A a 30.81Prochloraz 0.15 A d 0.14 A e 0.26 A d 0.46 A e 0.38 >100 a >100 a >100 a >100 a ndTebuconazole 38.75 A c 26.50 B d 1.70 C d 4.80 C d 3.87 >100 a >100 a >100 a >100 a ndThiophanate-methyl 35.25 A c 42.25 A c 3.42 B d 2.94 B de 22.65 >100 a >100 a >100 a >100 a ndThiram >100 A a >100 A a 18.75 C c 53.65 B b 6.19 1.04 B d 1.50 B b 0.49 C c 3.21 A b 0.49LSD(3)e 15.36 7.36 10.17 2.82 20.31 22.59 18.27 2.45

nd¼Not determined.a EC50 values (mg a.i. l�1).b Least significant difference: means followed by the same letter do not differ significantly (P< 0.05). Capital letters are for comparison of means in the same row. Small

letters are for comparison of means in the same column.c LSD0.05(1) is for comparison of means among pathogens with the same fungicide on mycelial growth.d LSD0.05(2) is for comparison of means among pathogens with the same fungicide on conidial germination.e LSD0.05(3) is for comparison of means among fungicides in the same pathogen on mycelial growth or conidial germination.

S. Alaniz et al. / Crop Protection 30 (2011) 489e494492

imazalil, prochloraz and thiophanate-methyl were the most effec-tive fungicides in inhibiting mycelial growth of C. macrodidymumisolates presenting EC50 values between 0.25 and 3.42 mg a.i. l�1.All the other fungicides, except tebuconazole for isolate Cy 14(EC50 value of 1.70 mg a.i. l�1), were significantly less effective ininhibiting mycelial growth, with EC50 values ranging from 4.80to >100 mg a.i. l�1.

3.1.2. Conidial germination assayAnalysis of variance showed that there were no significant

differences in the inhibition of conidial germination between thetwo conducted experiments, but the effect of fungicide, theirinteractions with pathogen and with pathogen isolates were allhighly significant (P< 0.001). Therewere also significant differencesbetween pathogen and pathogen isolates (P< 0.05) (Table 2). MeanEC50 values for reduction in conidial germination ofC. liriodendri andC. macrodidymum are given in Table 3. Captan, copper oxychloride,didecyldimethylammonium chloride and thiram were the mosteffective fungicides in reducing conidial germination of C. liriodendriisolates presenting EC50 values between 0.62 and 2.92 mg a.i. l�1,with the exception of copper oxychloride for the isolate Cy 59 (EC50value of 17.50 mg a.i. l�1). All the other fungicides were significantlyless effective in inhibiting conidial germination, with EC50 valuesranging from 6.65 to >100 mg a.i. l�1. Captan, copper oxychloride,cubiet, didecyldimethylammonium chloride and thiram were themost effective fungicides in reducing conidial germination ofC. macrodidymum isolates presenting EC50 values between 0.41 and4.45 mg a.i. l�1. All the other fungicides, were significantly lesseffective in inhibiting conidial germination, with EC50 valuesranging from 10.70 to >100 mg a.i. l�1.

3.2. Effect of selected fungicides on the rooting phase

Data of the two pot trials were combined because of the lack ofsignificant differences between the two experiments and itsinteraction with the studied variables (P> 0.05). With both path-ogens, P values indicated a significant effect of fungicide on RDSI

(P< 0.0001 and P< 0.0179 for C. liriodendri and C. macrodidymum,respectively) and percentage of reisolations (P¼ 0.0197 andP¼ 0.0492 for C. liriodendri and C. macrodidymum, respectively).The RDSI values and the percentages of reisolation of C. liriodendriand C. macrodidymum for each fungicide and control treatments areshown in Fig. 1.

All fungicides significantly decreased the RDSI values in bothspecies compared with control treatment, with the exception ofimazalil in C. macrodidymum. The RSDI values ranged from 1.0 to 2.0in the cuttings inoculated with C. liriodendri and from 1.12 to 1.76 inthe cuttings inoculated with C. macrodidymum. In the case of thepercentage of reisolation, all values were lower than those obtainedfor the control treatment, but only captan, carbendazim and dide-cyldimethylammonium chloride were significantly different in thecase of the cuttings inoculated with C. liriodendri, and prochloraz inthe case of those inoculated with C. macrodidymum. The percent-ages of reisolation ranged from 73.3 to 91.1% in C. liriodendri andfrom 70.5 to 88.5% in C. macrodidymum (Fig. 1).

4. Discussion

This and previous studies (Rego et al., 2006; Halleen et al., 2007;Rego et al., 2009) have shown the potential of several fungicides toreduce the risk of infection by C. liriodendri and C. macrodidymum,the causal agents of black foot disease of grapevines.

Results of the in vitro fungicide experiments were variabledepending on the method of evaluation used. In general, the activeingredients that showed a good response reducing mycelial growthdid not have a good performance inhibiting conidial germinationand most of the active ingredients that showed a good responseinhibiting conidial germination did not have a good performancereducing mycelial growth. Rego et al. (2006) obtained similarresults when these two methods were used to evaluate fungicidesto control C. liriodendri in vitro.

In agreement with our results, Rego et al. (2006) reported thatprochloraz was effective to reduce mycelial growth but was notable to inhibit conidial germination of C. liriodendri. They also

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Fig. 1. Effect of fungicides on root disease severity index (RDSI) and percentage of reisolation from roots of 110 R rootstock cuttings planted in substrate inoculated with Cylin-drocarpon liriodendri or C. macrodidymum for rooting. Vertical bars are the standard error of the means. The RDSI was analyzed with GENMOD procedure using the multinomialdistribution and the cumulative logit as link function, and means of the values were separated by chi-square test at P< 0.05. Analyses of variance (ANOVA) were conducted toanalyze the % of reisolation, and the values were separated LSD test at P< 0.05. Codes mean: Cap¼ captan, Car¼ carbendazim, Cop¼ copper oxychloride,Did¼ didecyldimethylammonium chloride, Hyd¼ hydroxyquinoline sulphate, Ima¼ imazalil and Pro¼ prochloraz.

S. Alaniz et al. / Crop Protection 30 (2011) 489e494 493

reported that tebuconazole was not effective to control bothmycelial growth and conidial germination of C. liriodendri. Some ofthe fungicides used in our experiments were also previously eval-uated in vitro by Halleen et al. (2007), but only to reduce mycelialgrowth of both C. liriodendri and C. macrodidymum. However, theresponses obtained by these authors were variable when comparedwith our results. In agreement with our experiment, imazalil andprochloraz were found effective to reduce the mycelial growth ofboth Cylindrocarpon species, but not the fungicide thiram. Contraryto our results, hydroxyquinoline sulphate was not able to reducethe mycelial growth of these pathogens. In the case of flusilazole,this fungicide reduced the mycelial growth of both species but, inour study, it was found effective only to reduce the mycelial growthof C. macrodidymum isolates.

Our results also illustrate the huge variation in fungicide sensi-tivity not only between species, but also within species. Furtherresearch is needed to investigate this effect and the potential offungicide mixtures to control Cylindrocarpon spp.

Chemical control can be used as a strategy to decrease the inci-dence and severity of Cylindrocarpon spp. during the nursery prop-agationprocess. In our study, seven fungicideswhich showed a goodperformance in vitro, were evaluated to control C. liriodendri andC. macrodidymum during the rooting phase. All fungicides were ableto decrease the RDSI values in inoculated grapevine rootstockcuttings compared with untreated controls. Nevertheless, thepercentages of reisolation of both pathogens from infected rootswere mostly not significantly different from those obtained in theuntreated controls. Therefore, the level of control achieved in thisphase was not sufficient to guarantee the production of healthygrapevine propagating material. Accordingly, Halleen et al. (2007)included prochloraz manganese chloride, benomyl, flusilazole andimazalil in semi-commercial field trials, but the incidence of blackfoot pathogens in grapevine plants was not significant and/orconsistent to be reduced by them. More satisfactory results wereobtained by Rego et al. (2009) when evaluating the effectiveness of

fludioxonil and cyprodinil and the mixtures of cyprodinil with flu-dioxonil and pyraclostrobin with metiram to prevent or reducenatural infections caused by Cylindrocarpon spp. in commercialnurseries. All fungicides except cyprodinil significantly reduced theincidence and severity of Cylindrocarpon spp.

All 14 fungicides included in our studywerepreviouslyevaluatedby Gramaje et al. (2009b) to control the Petri disease pathogensPhaeomoniella chlamydospora and Phaeoacremonium aleophilum ingrapevine nurseries in Spain. They found that soaking plantingmaterial in didecyldimethylammonium chloride during the hydra-tion stagewasone of themost effective treatments in limiting fungalinfection. These findings and those obtained in the present work,suggest that didecyldimethylammonium chloride could be includedin an integrated management program to control both Petri andblack foot diseases in grapevine nurseries. This product is notregistered for use in Spain, but has proven to be very effective toinhibit trunkdiseases pathogens in SouthAfrica grapevine nurseries(Fourie and Halleen, 2004b, 2006).

In addition, other control measures such as hot-water treat-ments can contribute to the management of black foot pathogens.Hot-water treatment protocols at 50 �C for 30 min are sufficient tocontrol Cylindrocarpon spp. in vitro (Bleach et al., 2009; Gramajeet al., 2010) and in vivo (Halleen et al., 2007). This treatment hasproved to be effective and should be recommended together withfungicide applications to improve black foot management ingrapevine nurseries.

Acknowledgements

This researchwasfinancially supported by the Projects TRT2006-11884-C04-01, RTA2007-00023-C04-03 (Programa Nacional deRecursos y Tecnologías Agrarias, Ministerio de Educación y Ciencia,Spain) and the European Regional Development Fund (ERDF). Weacknowledge V. Garrigues and A. Pedrero for technical assistance.

S. Alaniz et al. / Crop Protection 30 (2011) 489e494494

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