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Scientia Horticulturae 170 (2014) 53–60

Contents lists available at ScienceDirect

Scientia Horticulturae

journa l h om epage: www.elsev ier .com/ locate /sc ihor t i

valuation of the susceptibility of apple genotypes in Queretaro,exico to powdery mildew (Podosphaera leucotricha)

braham Paz-Cuadraa,1, Ludmila Elisa Guzmán-Pantojaa,1,amón Álvar Martínez-Penichea,∗,1, Coauthors Kruskaia Karenia Caltzontzin-Fernándezb,

uan Ramiro Pacheco-Aguilara, Sofía María Arvizu-Medranoa

División de Estudios de Posgrado, Facultad de Química, Universidad Autónoma de Querétaro, Centro Universitario s/n, Colonia las Campanas, 76010uerétaro, MexicoFacultad de Ciencias Naturales, Universidad Autónoma de Querétaro. Centro Universitario s/n, Colonia las Campanas, Querétaro 76010, Mexico

r t i c l e i n f o

rticle history:eceived 3 October 2013eceived in revised form 22 January 2014ccepted 24 January 2014vailable online 22 March 2014

eywords:enotypesusceptibilityowdery mildewreedingioassayorrelation

a b s t r a c t

The aim of this study was to evaluate the susceptibility to powdery mildew of apple (Malus domesticaBorkh) genotypes introduced in Queretaro, Mexico. For this study, a total of 38 apple genotypes were used:33 for the field assay and 14 for the greenhouse assay (with nine overlapping varieties). During the fieldassay, fungus incidence and severity were evaluated in adult plants grafted on the clonal rootstock MM106, and this test was performed on four consecutive evaluations from May to September 2012. On thelast sample, the conidial density was also determined on leaves with visible patches of powdery mildew.For the greenhouse bioassay, apple genotypes, grafted in January 2012 on MM 106, were inoculated ontheir adaxial side with 200 �L of 7 × 104 conidia/mL (14,000 conidia). The incidence and severity of thefungus on the new shoots were evaluated in August and in October. In addition, the conidial densitiesper cm2 of leaf area and per leaf were determined for the last sample, and the correlation between theparameters evaluated was examined in each assay and between assays. ‘Rayada’ and ‘411’ showed thehighest incidence (59.4 and 40.6%, respectively) and severity (120.0 and 128.3 mm2, respectively) topowdery mildew in the field bioassay, in which most genotypes showed no visible damage caused bythe fungus. ‘Lourdes’ and ‘436’ were the most resistant genotypes to powdery mildew in the greenhousebioassay (no incidence, or severity), which means that they are recommended for cultivation in humidenvironments, and/or where little agronomic management is undertaken. The situation was differentfor ‘424,’ ‘428’, and ‘Royal Gala’ (with incidence rates of 32.6, 27.5, and 25.5%, and severity rates of 86.6,

2

229.5 and 168.8 mm , respectively), which, given their agronomic and quality advantages, could be grownunder a strict phytosanitary program, or be used as an inoculum for experimental purposes. Significantcorrelations (P ≤ 0.05) between incidence and severity were obtained in most cases, but no significantcorrelation with respect to powdery mildew susceptibility under field and greenhouse conditions wasfound.

© 2014 Elsevier B.V. All rights reserved.

. Introduction

In the highlands of Central Mexico, in the State of Queretaro,exico, apples are grown without the use of irrigation (SIAP, 2011).

he main problems that hinder their growth are: the shortage ofdapted varieties to climate and the presence of fungal diseases,hich are poorly controlled, both of which lead to low yields, a

∗ Corresponding author. Tel.: +52 442 1921304; fax: +52 442 1921304.E-mail address: alvar@uaq.mx (R.Á. Martínez-Peniche).

1 These authors contributed equally to this work.

ttp://dx.doi.org/10.1016/j.scienta.2014.01.044304-4238/© 2014 Elsevier B.V. All rights reserved.

decrease in the commercial value of the produce and to a reductionof the useable surface area.

One of the most prevalent diseases in the region is powderymildew (Podosphaera leucotricha), which attacks plant foliage, pro-ducing white mycelia and gradually, leaf wrinkling; in more severecases, this disease can cause leaves and flowers to fall, and the fruitto develop russetting (Jones and Aldwinckle, 2002). These effectsare intensified with increases in temperature, solar radiation, and

moisture. Strong winds may also promote the dispersion of conidia(Sutton and Jones, 1979). Economic losses brought on by this dis-ease have been estimated to reach up to 50% (Jones and Aldwinckle,2002), and these losses tend to increase even further because new

5 a Horticulturae 170 (2014) 53–60

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Table 1Temperature, precipitation, and relative humidity in the experimental orchard dur-ing the period of evaluation.

Month T (◦C) P (mm) %HR

May 14.8 0.3 41.4June 15.4 5.1 58July 14.3 97.3 72.8August 14.4 168.7 73.0September 13.4 96.3 74.6

T: Average daily temperature, P: Monthly rainfall, HR: Average daily relative humid-ity.

Table 2Apple genotypes used in this study according to location.

Genotype Source Locationa

Field Greenhouse

‘401’ ‘Tropical Beauty’ × ‘Princess’ x‘403’ ‘Tropical Beauty’ × ‘Princess’ x‘406’ ‘Anna’ × ‘COOP30’ x‘407’ ‘Anna’ × ‘COOP30’ x‘411’ ‘Anna’ × ‘Gala’ x‘418’ ‘Anna’ × ‘CLR9T10’ x‘416’ ‘Anna’ × ‘CLR9T10’ x‘421’ ‘Anna’ × ‘Gala’ x‘419’ ‘Golden 650’ × ‘Gala’ x‘424’ ‘Anna’ × ‘Princess’ x x‘428’ ‘Anna’ × ‘Gala’ x x‘429-b’ ‘Anna’ × ‘CLR9T10’ x x‘436’ ‘Anna’ × ‘Gala’ x x‘438’ ‘Anna’ × ‘CLR9T10’ x‘441’ ‘Anna’ × ‘Gala’ x‘442–9’ ‘Anna’ × ‘CLR9T10’ x‘443’ ‘Einsherimer’ × ‘Princess’ x‘449’ ‘Anna’ × ‘CLR9T10’ x‘468’ ‘Anna’ × ‘Liberty’ x x‘SMA’ Unknown x‘SMB’ Unknown x‘SMC’ Unknown x‘SMD’ Unknown x‘SME’ Unknown x‘SMF’ Unknown x‘SMG’ Unknown x‘SM3’ Unknown x‘SM6’ Unknown x‘Lourdes’ Unknown x x‘Golden Delicious’ ‘Golden Reinette’ × ‘Grimes

Golden’x x

‘Delicious’ Selection in Madison, US x x‘Rayada’ Unknown, creole variety from

La Esperanza, Queretarox x

Malus micromalus Malus baccata × Malusspectabilis

x

‘Joya’ ‘Anna’ × ‘Gala’ x‘Royal Gala’ Mutant ‘Gala’ x‘Agua Nueva’ Mutant ‘Golden Delicious’ x‘Anna’ ‘Red Hadassiya’ × ‘Golden

Delicious’x

4 A. Paz-Cuadra et al. / Scienti

pple cultivars are generally highly sensitive to this fungus (Shol-erg et al., 2001).

Methods for controlling powdery mildew include: sanitaryanagement, which involves eliminating infected plant organs;

he use of agrochemicals, such as piperazines, pyridines, morpho-ines, and imidazoles (Jones and Aldwinckle, 2002), which can beetrimental to human health and the environment, and contributeo the development of resistant strains (Borlaug and Dowswell,002); biological control through the use of potentially antagonisticicroorganisms, such as Bacillus subtillis, Ampelomyces quisqualis,

nd Lecanicillium lecanni, which have been successful in control-ing Podosphaera fusca in melon (Romero et al., 2007); and the usef resistant varieties, an alternative that has proven to be the leastxpensive of all methods described above, and allows for profitableroductions in apple-growing regions, such as Queretaro, wherehytosanitary control is rarely practiced.

Resistance to powdery mildew of apple genotypes can be ana-yzed directly in the orchard, where plants are exposed to theatural environment and therefore, most likely exposed to severalhytopathogenic strains (Moore and Janick, 1998). Sholberg et al.2001), for example, conducted a four-year study on the powdery

ildew susceptibility of 13 varieties grafted on heavily infesteddult trees of ‘Jonagold.’ ‘Beesbe Delicious’ obtained the lowest inci-ence of conidia, compared to ‘Gala.’ Jones and Aldwinckle (2002)eported a high resistance to powdery mildew of ‘Delicious’ andYork Imperial’ under field conditions, compared to ‘Rome Beauty,’Granny Smith,’ and ‘Jonathan’.

The resistance to diseases, such as powdery mildew, can bessessed in greenhouses, and given the more controlled envi-onment of greenhouses compared to the open field, a greaternoculum pressure can be exerted. Blazek (2004) evaluated theusceptibility of apple seedlings to powdery mildew, by sprayingonidial solutions on young shoots; those showing the highest tol-rance in the greenhouse were then evaluated over the course of0 years in the field. Jeger et al. (1986) reported differences inhe susceptibility of several apple cultivars inoculated with a fixedoncentration of spores; ‘Discovery’ proved to be highly resistant,Bramley’s Seedling’ and ‘Suntan’ were resistant and ‘Golden Deli-ious,’ very susceptible.

With respect to the availability of apple types within the region,ow-chilling varieties, most of which had been bred in Northern

exico, were introduced to Queretaro, and were evaluated in termsf their flowering and ripening seasons, yield, and quality (Mendozat al., 2008). Although a few outstanding genetic types have beenetected, their susceptibility to the main diseases prevalent in theegion remains unknown.

The aim of this study was to evaluate the susceptibility toowdery mildew of different apple genotypes, which have been

ntroduced in Queretaro, through assays conducted under field andreenhouse conditions, and to determine the correlation betweenhese two methods.

. Materials and methods

.1. Experiment locations and plant material

Firstly, the in-field study was performed within a non-irrigatedrchard, located in the municipality of Cadereyta, in the State ofueretaro, Mexico, situated at 20◦51′ NL, 99◦35′ WL and at an eleva-

ion of 2495 m. The annual average temperature and precipitationre 12.6 ◦C and 812.5 mm, respectively.

The climatic conditions recorded in the orchard during the eval-ation period are shown in Table 1 (CEA, 2012).

Secondly, the in-greenhouse study was carried out at theutonomous University of Queretaro (UAQ), in the State of

‘Rosada’ Unknown, creole variety fromAmealco, Queretaro

x

a Site where the genotype was evaluated.

Queretaro, located at 20◦36′ NL, 99◦34′ WL, at an elevation of1822 m. The temperature and relative humidity inside the green-house fluctuated between 17 to 25 ◦C and 30 to 69%, respectively.

A total of 38 apple genotypes were used, 33 for the field assayand 14 for the greenhouse assay (with nine overlapping varieties),and consisting of several cultivars, creoles varieties, mutants, andhybrids (Table 2).

2.2. Identification and preservation of P. leucotricha

The inoculum was obtained from visible growth patches onapple leaves, collected from the orchard chosen for the experiment.

A. Paz-Cuadra et al. / Scientia Horticulturae 170 (2014) 53–60 55

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Fig. 1. Images of P. leucotrichia obtained from apple leaves.

n order to discern the fungus, its typical structures were identifiedhrough a microscope using thin sections of infected tissuesFig. 1). In order to preserve the samples for their subsequentse in-greenhouse bioassays, the fungus was inoculated in plantslaced in pots.

.3. Preparation of the inoculum

Leaves infested with the fungus were immersed in 20 mL ofterile distilled water, which was gently shaken for 1 min; theupernatant containing the spores was placed in a flask and thepore concentration was calculated using a Neubauer hematocy-ometer (depth: 0.1 mm). The stock solution was mixed with 2%lycerol and stored at −20 ◦C.

.4. Field bioassay

Genotypes to be evaluated were introduced in the orchard byrafting them onto adult plants of ‘Golden Delicious’ / MM 106 in001. Four branches were randomly selected for each genotype,nd subsequently, 10 leaves of each branch were evaluated for: (a)he incidence of powdery mildew (percentage of leaves with visibleresence of powdery mildew on the adaxial and/or abaxial sides)nd; (b) the severity of the fungus (leaf area covered by fungus, cal-ulated by measuring the length and width of the lesion) (Sholbergt al., 2001). This test was performed on four consecutive samplesrom May to September 2012.

On the last sample, the conidial density was also determined oneaves with visible patches of powdery mildew. The leaves werelaced in a flask with 10 mL sterile distilled water and the flaskas shaken (100 rpm during 30 min). An aliquot was then sampled

rom the obtained solution in order to calculate the total number of

onidia, using a Neubauer hematocytometer, and subsequently, theonidial density (Sholberg et al., 2001), through the determinationf the leaf area by means of image analysis using the MATLAB®

oftware (Vázquez-Cruz et al., 2012).

maged young shoots. (b) Conidial chains (4×). (c). (100×).

2.5. Greenhouse bioassay

Apple genotypes were grafted in January 2012 on a clonal root-stock MM 106, which was placed in a pot. In May of that year, each ofthe first 10 fully-expanded, upper leaves of six plants of each geno-type were inoculated on their adaxial side with 200 �L of 7 × 104

conidia/mL (14,000 conidia). The incidence and severity of the fun-gus on the new shoots, which were exposed to natural inoculumspresent in the greenhouse, were measured on two occasions, firstin August and then in October. The conidial densities per cm2 of leafarea and per leaf were also determined for the last sample (Sholberget al., 2001).

2.6. Data analysis

Analysis of variance and the Tukey test were applied to the dataobtained. The incidence and severity were analyzed as a bifactorialdesign, being the factors the genotypes and sampled periods, andalso for each sample period. For the field experiment, only thosegenotypes showing visible damage caused by the fungus were ana-lyzed. As the sampling distribution of parameters on the naturalscale is non-normal, incidence values were converted to angulardegrees [Arc sine

√(% reported/100)], the severity value was con-

verted to√

(x + 1), and the conidial density was converted to log10(x + 1). The correlation between the parameters evaluated was alsoexamined in each assay and between assays. JMP statistical soft-ware version 8.0 (SAS Institute, 2005) was employed for these dataanalyses.

3. Results

3.1. Field evaluation

3.1.1. IncidenceOnly 13 of the 33 evaluated genotypes exhibited visible symp-

toms of powdery mildew. They also showed different percentages

56 A. Paz-Cuadra et al. / Scientia Horticulturae 170 (2014) 53–60

Table 3Incidence rates of P. leucotricha on the leaves of 13 apple genotypes examined on four different periods under natural inoculum in-field conditions.

Genotype Period Averages by genotype

May 11th June 7th July 4th September 10th

‘Rayada’ 50 (±0)a a 70 (±4.1) a 57.5 (±4.8) a 60 (±5.8) a 59.4 (±4.1) a‘411’ 32.5 (±7.5) ab 35 (±5) ab 47.5 (±2.5) a 47.5 (±2.5) a 40.6 (±4) b‘429-b’ 7.5 (±2.5) cd 12.5 (±4.8) bc 15 (±6.4) bc 15 (±5) b 12.5 (±1.8) c‘SM6’ 15 (±5) bc 30 (±0) b 0 (±0) bc 0 (±0) c 11.2 (±7.2) cd‘449’ 10 (±0) bcd 10 (±0) bc 10 (±0) bc 10 (±0) b 10 (±0) c‘438’ 10 (±0) bcd 10 (±0) bc 7.5 (±2.5) bc 10 (±0) b 9.4 (±0.6) c‘403’ 5 (±2.9) cd 5 (±2.9) c 12.5 (±2.5) b 12.5 (±2.5) b 8.7 (±2.2) cd‘424’ 5 (±2.9) cd 15 (±11.9) bc 15(±11.9) bc 0 (±0) c 8.7 (±3.7) cd‘SMA’ 0 (±0) d 7.5 (±2.5) bc 12.5 (±2.5) b 12.5 (±2.5) b 8.1 (±2.9) cd‘418’ 10 (±0) bcd 10 (±0) bc 10 (±0) bc 0 (±0) c 7.5 (±2.5) cd‘SMC’ 2.5 (±2.5) cd 7.5 (±2.5) bc 10 (±0) c 10 (±0) b 7.5 (±1.8) cd‘SMB’ 0 (±0) d 7.5 (±2.5) bc 10 (±0) bc 10 (±0) b 6.9 (±2.4) cd‘Golden D’ 7.5 (±2.5) cd 10 (±0) bc 0 (±0) c 0 (±0) c 4.4 (±2.6) d“Fvalue genotypes 11.0** 8.4** 12.1** 50.0** 42.5**

Tukey DMS 19.1 22.3 19.8 11.2 8.9“Fvalues period 6.5**

Averages by period 11.9 c 17.7 a 16.0 ab 14.4 bc 15.0“Fvalues (genotype × period) 4.3**

The averages followed by different letters indicate the statistical differences (Tukey, 0.05).tition

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a Average percentage of incidences obtained from 40 individual results (four repeegrees (arcsine

√%I/100) for their analysis and they are included in the table in ter

** Highly significant differences (P ≤ 0.01).

n pathogen incidence, even in each of the four evaluations: ‘Rayada’nd ‘411’ (‘Anna’ × ‘Gala’) obtained the highest values (59.4 and0.6%, respectively). The other genotypes, including ‘Golden Deli-ious’ and ‘403’ (‘Tropical Beauty’ × ‘Princess’), presented incidenceercentages lower than 12.5% (Table 3).

The lowest pathogen incidence with visible symptoms of theisease, occurred on May 11th (X = 11.9%). The interaction (geno-ype × period) was also significant (“F” = 4.3**)

.1.2. Severity and number of conidiaOnce again, ‘Rayada’ and ‘411’ were the most susceptible geno-

ypes, demonstrating the highest severity average values (120.0nd 128.3 mm2, respectively), with no differences in comparisono ‘438 (56.2 mm2), but both are higher than other genotypes,hile the highest number of conidia per cm2 of leaf surface area

able 4everity (mm2) of P. leucotricha on leaves of 13 apple genotypes, evaluated in four period

Genotype Period

June 7th July 4th

‘411’ 40.5 (±6.4)a ab 143.8 (±53.1) a

‘Rayada’ 49.3 (±2.2) ab 106.6 (±42) ab

‘438’ 64.5 (±25.5) ab 53.9 (±15.3) abc

‘403’ 39.7 (±23) ab 30.9 (±39.7) bcd

‘SMA’ 22.5 (±9.2) ab 23.8 (±2.8) bcde‘SMB’ 28 (±12.9) ab 31.9 (±8.6) bcde‘SMC’ 28.3 (±12.1) ab 16.8 (±2.8) cdef

‘429-b’ 34 (±12.8) ab 6.4 (±3.1) def

‘Golden D’ 82.2 (±42.3) a 0 (±0) f

‘449’ 11.6 (±4.4) ab 2.1 (±0.4) def

‘SM6’ 48.8 (±14.2) ab 0 (±0) f

‘418’ 7.8(±2.5) ab 8.2 (±3) cdef

‘424’ 0.7 (±0.5) b 0.8 (±0.5) ef

“F” values genotypes 2.2* 13.1**

Tukey DMS 7.2 5.0

”F” value period 1.6NS

Averages by period (NS) 35.3 a 33.4 a

“F” value (genotype × period) 4.0**

he averages followed by different letters indicate the statistical differences (Tukey, 0.05a Average severity (mm2) obtained from 40 individual results (four repetitions with 10 l

og10(x + 1), respectively, for their analysis and were included in the table in terms of the

ifference.* Significant differences at P ≤ 0.05.

** Significant differences at P ≤ 0.01.

s with 10 leaves in each) and standard error. The results were converted to angular the original parameter. DMS: minimum significant difference.

(nc/cm2) was obtained by ‘411’ (563.3 × 104 nc/cm2), followedby ‘SMC’ and ‘449’ (113.1 and 109.4 × 104 nc/cm2, respectively)(Table 4). No differences in periods are revealed, but interaction(genotype × period) is again present.

3.1.3. Correlation between incidence, severity and number ofconidia

The correlation coefficients between the observed incidences inthe different periods (1st quadrant) were higher than 0.817**. Withregard to severity (4th quadrant), significant correlations were also

obtained, except for those related to the sample conducted in June(Table 5).

In terms of the correlation between the incidence and severityrates of the fungus (3rd quadrant), excluding the results obtained

s and the number of conidia/cm2, under natural inoculum in-field conditions.

Averages by genotype (1 × 104) nc/cm

September 10th

200.5 (±65.2) a 128.3 (±40.5) a 563.3 a204 (±80.1) ab 120.0 (±39.1) a 19.2 bc50.2 (±14.4) c 56.2 (±3.7) ab 50.3 bc57.2 (±6.3) bc 42.7 (±6.7) bc 3.7 c

f 73.6 (±29) abc 40.0 (±14.6) bc 19.3 bc 41.5 (±14.4) cd 33.8 (±3.5) bcd 17 bc

16.8 (±2.8) cd 20.6 (±3.3) bcde 113.1 ab9.1 (±4.4) cd 16.5 (±7.6) cdef 13.9 bc0 (±0) d 27.4 (±3.7) cdef 0 d13.2 (±4.7) d 9.0 (±3) cdef 109.4 ab0 (±0) d 16.3 (±14.1) def 0 d0 (±0) d 5.3 (±2.3) ef 0 d0 (±0) d 0.5 (±0.2) f 0 d15.1** 18.5** 162.2**

6.1 3.4 1.0

51.2 a 39.7

). NS: not significant.eaves in each) and standard error. Severity and conidial data were converted to andoriginal parameter. n: number of conidia/cm2 leaf area. DMS: minimum significant

A. Paz-Cuadra et al. / Scientia Horticulturae 170 (2014) 53–60 57

Table 5Correlation coefficients of incidence, severity and number of conidia of Podosphaera leucotricha on leaves of different apple genotypes observed during the in-field study.

Variable %Ip May %Ip June %Ip July %Ip September %Ip over time Sp June Sp July Sp September Sp over time (1 × 104) nc/cm2

%Ip May 1.000%Ip June 0.956** 1.000%Ip July 0.871** 0.827** 1.000%Ip September 0.870** 0.817** 0.963** 1.000%Ip over time 0.965** 0.940** 0.954** 0.950** 1.000Sp June 0.328NS 0.302NS 0.097NS 0.241NS 0.248NS 1.000Sp July 0.777** 0.676** 0.877** 0.905** 0.856** 0.300NS 1.000Sp September 0.817** 0.755** 0.927** 0.959** 0.915** 0.260NS 0.960** 1.000Sp over time 0.815** 0.738** 0.869** 0.925** 0.884** 0.454NS 0.972** 0.973** 1.000(1 x 104) nc/cm2 0.415NS 0.259NS 0.541* 0.545* 0.467NS 0.070NS 0.726** 0.606** 0.625* 1.000

* Significant correlation at P ≤ 0.05.** Significant correlation at P ≤ 0.01.

Table 6Incidence of Podosphaera leucotricha on leaves of 14 apple genotypes evaluated in two periods examined in greenhouse with artificial inoculum.

Genotype Period Averages by genotype

13th August 9th October

‘424’ 33.7 (±11.8)a a 31.4 (±13.2) a 32.6 (±1.2) a‘428’ 25 (±15) ab 30 (±20) a 27.5 (±2.5) abc‘Royal Gala’ 32.5 (±11.1) ab 18.6 (±8.4) a 25.5 (±7) ab‘Joya’ 23.3 (±14.1) ab 23.3 (±14.1) a 23.3 (±0) abc‘Rayada’ 20 (±5.5) ab 22 (±5.8) a 21 (±1) abc‘429-b’ 3.3 (±3.3) ab 26.7 (±26.7) a 15 (±11.7) abc‘Anna’ 5 (±5) ab 13.3 (±6.1) a 9.2 (±4.2) abc‘Agua Nueva’ 8.6 (±7) ab 8.6 (±7.4) a 8.6 (±0) bc‘Golden D’ 6.7 (±4.9) ab 1.7 (±1.7) a 4.2 (±2.5) bc‘Delicious’ 2.8 (±2.8) b 4.3 (±3) a 3.6 (±0.7) bc‘Rosada’ 3.3 (±3.3) ab 3.3 (±3.3) a 3.3 (±0) abc‘468’ 0 (±0) b 3.3 (±3.3) a 1.7 (±1.7) bc‘436’ 0 (±0) b 0 (±0) a 0 (±0) c‘Lourdes’ 0 (±0) b 0 (±0) a 0 (±0) c“F” Values genotypes 3.3** 1.8 NS 4.7**

Tukey DMS 38.2 43.1 26.6“F” value periods 0.14NS

Averages by period 11.7 a 13.3 a 12.5“F” value (genotype × period 0.4NS

The averages followed by different letters indicate the statistical differences (Tukey, 0.05). NS: not significant.a Average incidence percentage, obtained from 30 individuals results (three repetitions with 10 leaves in each) and standard error. The results were converted to angular

degrees (arcsine√

%I/100) for their analysis and included in the table in terms of the original parameter. : average incidence. DMS: minimally significant difference.** Highly significant differences (P ≤ 0.01).

Table 7Severity (mm2) of P. leucotricha on leaves of 14 apple genotypes evaluated in two periods, the number of conidia/cm2 and the number of conidia/leaf, examined in greenhousewith artificial inoculum.

Genotype Period Averages by genotype (1 × 104)nc/cm2

(1 × 104) nc/leaf

13 August 9 October

‘428’ 180.3 (±84.2)a a 278.8 (±211.2) a 229.5 (± 49.2) a 153.1 a 715.5 a‘Royal Gala’ 240.0 (±117.5) a 97.2 (±36.9) ab 168.6 (±71.4) a 48.8 abc 117.2 a‘424’ 71.1 (±37.6) ab 102 (±48.1) ab 86.6 (±15.5) ab 557.5 a 517.5 a‘Joya’ 52.5 (±28) ab 65.4 (±32) ab 58.9 (±6.5) ab 10 abc 69.4 a‘Rayada’ 28.8 (±13.5) ab 55.7 (±18.9) ab 42.3 (±13.4) ab 16.2 abc 75.0 a‘Anna’ 8.1 (±8.12) b 58.9 (±35.9) ab 33.5 (±25.4) b 334.4 a 221.9 a‘Agua Nueva’ 34.1 (±31.6) b 26 (±23.5) ab 30.1 (±4) b 7.0 c 21.3 a‘Golden D’ 24.0 (±15.7) b 12.7 (±12.7) b 18.4 (±5.7) b 5.9 abc 275.0 a‘468’ 0 (±0) b 24.8 (±24.8) ab 12.4 (±12.4) b 143.2 ab 169.8 a‘Rosada’ 1.1(±1.1) b 18.8 (±18.8) ab 10 (±8.8) b 0.4 c 25.0 a‘429-b’ 3.5 (±3.5) b 12.0 (±12) b 7.8 (±4.3) b 24.7 abc 25.0 a‘Delicious’ 1.8 (±1.8) b 5.1 (±4.1) b 3.5 (±1.6) b 2.3 bc 37.5 ab‘436’ 0 (±0) b 0 (±0) b 0 (±0) b 0 d 0 b‘Lourdes’ 0 (±0) b 0 (±0) b 0 (±0) b 0 d 0 b“F” values genotypes 4.2** 2.2* 5.5** 46.3** 8.4Tukey DMS 9.4 11.4 6.9 5.2 4.0“F” values periods 0.4NS

Averages by period 46.1 a 54.1 a 50.1“F” value (genotype × period) 0.8NS

The averages followed by different letters indicate the statistical differences (Tukey, 0.05). NS: not significant.a Average severity (mm2) obtained from 30 individuals results (three repetitions with 10 leaves in each) and standard error. Severity and conidial data were converted to

and log10(x + 1) for their analysis and included in the table in terms of the original parameter. n: number of conidia. : average severity. DMS: minimally significant difference.* Significant differences at P ≤ 0.05.

** Significant differences atand P ≤ 0.01.

58 A. Paz-Cuadra et al. / Scientia Horticulturae 170 (2014) 53–60

Table 8Correlation coefficients and significance in terms of incidence and severity rates and the number of conidia of Podosphaera leucotricha in apple leaves, observed during thein-greenhouse analysis.

Variable %Ip August %IpOctober

%Ip over time Sp August Sp October Sp over time (1 x 104) nc/cm2 (1 x 104) nc/leaf

%Ip August 1.000%Ip October 0.778* 1.000%Ip Over time 0.946** 0.939** 1.000Sp August 0.791** 0.525NS 0.702** 1.000Sp October 0.680** 0.679* 0.721** 0.751** 1.000Sp Over time 0.787** 0.643* 0.760** 0.936** 0.935** 1.000(1 × 104) nc/cm2 0.270NS 0.392NS 0.349NS 0.149NS 0.433NS 0.310NS 1.000(1 × 104) nc/leaf 0.551* 0.554* 0.586* 0.509* 0.847** 0.724** 0.625* 1.000

Average percentage of incidence (%Ip) and average severity (Sp) of powdery mildew on apple leaves under greenhouse conditions. The analysis was performed with 14g

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ripening) and ‘436’ (middle ripening). It is unlikely that the absenceof symptoms of powdery mildew in this group was geneticallyrelated; it could be attributed, for instance, to an inadequate naturalinoculation of the plants in the orchard. The inoculum level tends

Table 9Correlation coefficients for incidence and severity rates and the number of conidiaof Podosphaera leucotricha in apple leaves under field versus greenhouse conditions.

Variable %Ip over timegreenhouse

Sp over timegreenhouse

(1 × 104) nc/cm2

greenhouse

%Ip over time,field

0.372NS −0.013NS −0.133NS

Sp over time,field

0.243NS −0.066NS −0.282NS

(1 x 104)nc/cm2, field

0.298NS −0.124NS −0.279NS

enotypes. nc/cm2: number of conidia per cm2 of leaf area. NS: not significant.* Significant correlation at P ≤ 0.05

** Significant correlation at P ≤ 0.01.

or the severity recorded in June, r values were always higher than.676**.

Finally, nc/cm2 significantly correlated with both pathogenncidence (5◦ quadrant) and severity (6◦ quadrant) in July andeptember.

.2. Greenhouse evaluation

.2.1. IncidenceDifferences in the percentage of incidence for powdery mildew

P ≤ 0.001) between genotypes were detected (Table 6): genotypes424’ and ‘428’ (both early ripening) showed the highest values32.6 and 27.5%), but with no statistical differences when comparedo a group of early ripening genotypes, including ‘Royal Gala,’ ‘Joya,’Rayada,’ and ‘Agua Nueva’ (which is a mutant variety of ‘Goldenelicious’). ‘Golden Delicious’ and ‘Delicious’ show comparable lev-ls of incidence. Finally, ‘436’ and ‘Lourdes’, no damage caused byhe pathogen was detected, in either field or greenhouse studies.

.2.2. Severity and number of conidiaThe highest average severity rate occurred for ‘428’

229.5 mm2), (4th column), with no significant differences withRoyal Gala,’ ‘424,’ ‘Joya’, 429-b and ‘Rayada’ (168.6, 86.6, and 58.9nd 42.3 mm2, respectively) but they were higher than any otherarieties, while the highest number of conidia per cm2 of leaf areanc/cm2) was achieved by ‘424’ (557.5 × 104 nc/cm2), followedy ‘Anna’ and ‘428’ (334.4 and 153.1 × 104 nc/cm2, respectively).n the other side of the spectrum, no symptoms of damage wereetected for ‘436’ and ‘Lourdes.’ The lowest number of conidia per

eaf (nc/leaf) was also obtained by these two genotypes (Table 7).

.2.3. Correlation between incidence, severity, and number ofonidia

All correlation coefficients between fungus incidences at dif-erent times (1st quadrant) and severities at different times (4thuadrant) were highly significant. In addition, the coefficientsetween incidences and severities (3rd quadrant) were also sig-ificant in most cases (Table 8).

Contrary to the results of the tests conducted in field, no sig-ificant correlation between the nc/cm2 and the incidence or theeverity were detected in any of the periods analyzed, but there was

correlation between the nc/leaf ratio and both of these variables,n both periods (quadrants 5 and 6).

.3. Correlation of powdery mildew susceptibility in field and

reenhouse conditions

No significant correlation in terms of the susceptibility toowdery mildew of the genotypes evaluated under field and

greenhouse conditions, for any of the three variables considered,were found (Table 9).

The highest correlation coefficient was obtained between theaverage in-field and in-greenhouse incidence rates (r = 0.372NS).The results for the corresponding regression analysis are shown inFig. 2, where ‘Lourdes’, ‘436’, ‘468’, ‘Delicious’, and ‘Golden Deli-cious’ proved to be relatively tolerant to the pathogen in bothenvironments. The opposite is illustrated by ‘Rayada’, which is sus-ceptible to the pathogen under both conditions. However, ‘428’,‘424’, and, to a lesser extent ‘429-b’, showed little or no presenceof the pathogen when evaluated in field, contrasting with theirbehavior exhibited under greenhouse conditions.

4. Discussion

4.1. Field evaluation

The highest values of incidence and severity were obtained by‘Rayada’ and ‘411’. The first one is a Creole variety introduced fromthe USA, with great local appeal since it blooms late, ripens earlyand produces a very attractive red fruit (Mendoza et al., 2008),while ‘Gala,’ one of the progenitors of ‘411,’ is considered suscep-tible to powdery mildew (Sholberg et al., 2001). ‘Golden Delicious’and ‘403’ (‘Tropical Beauty’ × ‘Princess’) presented lower incidencepercentages. The first one and ‘Princess’ are also susceptible topowdery mildew (Biggs et al., 2009).

The 20 genotypes that presented no symptoms of powderymildew during the experiment include ‘Delicious,’ considered mod-erately resistant (Korban and Riemer, 1990), Malus micromalus andthe promising red varieties ‘Lourdes’ (late ripening), ‘428’ (early

Average percentage of incidence (%Ip) and average severity (Sp) of powdery mildewon apple leaves evaluated under greenhouse and field conditions. The analysis wasconducted using the same nine genotypes under both conditions. N: Number ofconidia. NS: not significant.

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ig. 2. Linear regression analysis of the incidence rate of powdery mildew on applesnder field versus greenhouse conditions.

o be low when ecological factors are unfavorable, naturally lead-ng to a low incidence of the fungus (Moore and Janick, 1998). Theverage temperature during the evaluation period was 14.5 ◦C, theelative humidity was around 61.7%, and the average monthly pre-ipitation was only 49.7 mm (Table 2), however, this fungus thrivesuring warm and humid summer months, and withers when theelative humidity is lower than 70% (Jones and Aldwinckle, 2002).

The fact that the lowest pathogen incidence occurred on May1th is probably due to the fact the RH was lower than on otheronths (41.4%) (Table 1), an unfavorable condition for the prolif-

ration of the pathogen (Sutton and Jones, 1979). The significantnteractions (genotype × period) (Tables 3 and 4) were certainlyue to the major incidence or severity of ‘SM6’ and ‘Golden Deli-ious’ in June the 7th.

The high correlation coefficients between the observed inci-ences and between the severities in the different periods implyhat susceptibility to the pathogen of the genotypes was generallyonsistent throughout the period of evaluation, and the correlationetween the incidence and severity rates of the fungus coincidesith the report published by Sholberg et al. (2001).

Finally, the significant correlations between number ofonidia/cm2 leaf area (nc/cm2) with both pathogen incidence andeverity in July and September (month in which the conidia wereounted) agree with Sholberg et al. (2001) who state that appleenotypes with high severity of powdery mildew show high den-ities of conidia on the leaves. Moreover, Dewdney et al. (2003)eported that the concentration of conidia of Venturia inaequalis onhe leaves is correlated with the susceptibility to this pathogen.

.2. Greenhouse evaluation

Genotypes showing the highest incidence to oidium included424’ and ‘428’, both early ripening (Mendoza et al., 2008), ‘Royalala,’ considered by Sholberg et al. (2001) as being very suscepti-le to powdery mildew in studies realized in two sites over fourears, ‘Joya,’ ‘Rayada,’ and ‘Agua Nueva’, which is a mutant vari-ty of ‘Golden Delicious’ (Ramírez, 1986). In the case of ‘Rayada,’ itas been recommended that this genotype be introduced into theegion, and although it presents agronomic and quality advantages,iven its sensitivity to this fungus, both in field and in green-ouse, as shown in this study, a chemical control program woulde required. The comparable levels of susceptibility obtained by

Golden Delicious’ and ‘Delicious’ contrast with those reported byanick et al. (1996) and Biggs et al. (2009). Finally, ‘436’ and ‘Lour-es’ which showed no damage caused by the pathogen, in either

eld or greenhouse studies are good quality intermediate and lateipening genotypes, respectively (Mendoza et al., 2008).

Contrary to the field evaluation, the differences in the suscep-ibility to powdery mildew can be considered genetic, which is

iculturae 170 (2014) 53–60 59

perhaps related to the capacity to synthesize resistance metabo-lites (Hernández et al., 2002), since all materials were evaluatedin the same environmental conditions and under a high inoculumpressure within the greenhouse.

Therefore, we could classify the genotypes as: resistant, slightlysusceptible, moderately susceptible, and highly susceptible. Firstly,the resistant genotypes (with no visible symptoms of the fungus)include: ‘Lourdes’ and ‘436’. These genotypes could be recom-mended for cultivation in climates that favor the development ofthe pathogen, and/or where phytosanitary control is practiced fromthe outset. They also possess appealing local agronomic and qualitycharacteristics. ‘Lourdes’ is a late ripening cultivar, produces attrac-tive fruit and presents high storage ability; ‘436’ produces a big redfruit and presents a high yield (Mendoza et al., 2008). Secondly,slightly susceptible genotypes (with moderate fungus growth, from3.5 to 40 mm2), include: ‘Delicious’, ‘429-b’, ‘Rosada’, ‘468’, ‘GoldenDelicious’, ‘Agua Nueva’ and ‘Anna’. Korban and Riemer (1990) havedeemed ‘Delicious’ as moderately resistant and ‘Golden Delicious’as susceptible to powdery mildew. Thirdly, the moderately sus-ceptible genotypes (with significant mildew growth, from 41 to80 mm2) include: ‘Joya’ and ‘Rayada’. And finally, the most suscep-tible (massive colonization, covering the entire leaf area, ≥81 mm2)include: ‘424,’ ‘Royal Gala,’ and ‘428,’ which could be used as asource of inoculums for the evaluation of the susceptibility to pow-dery mildew of new genetic materials, both in greenhouse or invitro. However, recommendations for their introduction and cul-tivation in the region should take into consideration their highsusceptibility to powdery mildew.

The highly significant correlation coefficients between fun-gus incidences and between severities confirm that sensitivityto the pathogen among the genotypes is consistent over time.In addition, the significant coefficients between incidences andseverities in most cases also confirm the results obtained in thefield.

Finally, no significant correlations between the nc/cm2 and theincidence or the severity detected contrast with the results of thetests conducted in field. The nc/cm2 has been used to estimate thesusceptibility to powdery mildew in apples (Sholberg et al., 2001),cherries, grapes (Sholberg and Boulé, 2009) and in apple scabs(Dewdney et al., 2003). But the correlation between the nc/leaf ratioand both of these variables, in both periods is logic since nc/leafratio takes into account the total conidia produced, regardless ofthe surface area of the leaves.

4.3. Correlation of powdery mildew susceptibility in field andgreenhouse conditions

The absence of significant correlation in terms of the suscepti-bility to powdery mildew of the genotypes evaluated under fieldand greenhouse conditions, for any of the three variables consid-ered coincides with Jeger et al. (1986), who found that the relativeseverity of this fungus in ‘Crispin’ and ‘Spartan’ was inconsistentwhen analyzed under field and greenhouse conditions. Sholberget al. (2001) reported that the range of genetic susceptibility ofdifferent genotypes usually changes in relation to the particularabiotic factors of the experimental site and to the year in whichthe assessment is conducted. Other authors, such as Jones andAldwinckle (2002), Moore and Janick (1998), and Biggs et al. (2009),have reported that there is no behavioral connection in relationto powdery mildew across the different genotypes when they areexposed to different environmental conditions. In our case, the lowcorrelation coefficient obtained between the average in-field and

in-greenhouse incidence rates (r = 0.372NS) (Fig. 2) is due to thelack of correspondence in the susceptibility to powdery mildew of‘428’, ‘424’, and, to a lesser extent ‘429-b’, in the field and undergreenhouse conditions.

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. Conclusions

. ‘Rayada’ and ‘411’ showed the highest sensitivity to powderymildew in the field bioassay, in which most genotypes showedno visible damage caused by the fungus.

. ‘Lourdes,’ ‘436,’ and ‘468’ were the most resistant genotypes topowdery mildew in the greenhouse bioassay, which means thatthey are recommended for cultivation in humid environments,and/or where little agronomic management is undertaken. Thesituation is different for ‘424,’ ‘Royal Gala,’ and ‘428,’ which, giventheir agronomic and quality advantages, could be grown undera strict phytosanitary program, or be used as an inoculum forexperimental purposes.

. No significant correlation with respect to powdery mildew sus-ceptibility under field and greenhouse conditions among thevarious genotypes evaluated was found.

ppendix A. Supplementary data

Supplementary data associated with this article can beound, in the online version, at http://dx.doi.org/10.1016/j.scienta.014.01.044http://dx.doi.org/10.1016/j.scienta.2014.01.044.

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