We thank Andrea Patocchi, Muhammad Awais Khan, Giovanni Broggini and Hans Jansen for helpful support in QTL analysis; Markus Kellerhals, Mauro Jermini ...
Apple resistance to arthropod herbivores: genetic basis and modification by environmental factors Karsten Mody1, Sibylle Stoeckli1, Cesare Gessler2, Silvia Dorn1 1 ETH Zurich, Institute of Plant Sciences / Applied Entomology, 8092 Zurich, Switzerland 2 ETH Zurich, Institute of Integrated Biology / Plant Pathology, 8092 Zurich, Switzerland Abstract: Arthropod herbivores reduce the quantity and quality of apple yield. Resistant apple varieties hold promise to increase the sustainability of pest management in orchards, but little is known on the genetic basis of apple resistance to most arthropod herbivores. Knowledge on the apple genome and QTL (quantitative trait locus) analysis is now facilitating the identification of gene regions associated with resistance. 160 F1-progeny plants of a cross of the apple varieties 'Fiesta' and 'Discovery' were surveyed at three different sites in Switzerland. Herbivore infestation per genotype as a measure of resistance was quantified for the apple aphids Dysaphis plantaginea, Dysaphis cf. devecta and Aphis pomi, the apple rust mite Aculus schlechtendali, and the codling moth Cydia pomonella. The influence of the environmental factor 'drought stress' on apple resistance to a chewing and a sap-feeding herbivore (caterpillar; aphid) was studied in laboratory experiments considering different intensities of pulsed drought stress. Significant QTLs for resistance to D. plantaginea, D. cf. devecta, A. schlechtendali, and C. pomonella were detected. SSR alleles associated to the QTLs may be applied to identify and breed resistant apple cultivars. Environmental factors such as within-canopy variation in climate, and neighbourhood-effects affected herbivore distribution in the field. In the laboratory, pulsed drought stress resulted in non-monotonic resistance responses of apple trees. Low-stress plants showed the highest and high-stress plants the lowest resistance. The studies revealed the genetic basis of apple resistance to different arthropod herbivores and the modifying influence of environmental parameters that may impede QTL detection. Key words: Apple Malus x domestica, aphids, caterpillars, deficit irrigation, host plant resistance, individual tree genotypes, interactions, pest management, QTLs, pulsed drought stress
Introduction Apple (Malus x domestica Borkh.) is the most relevant fruit crop in the temperate region. Arthropod herbivores have a negative impact on the quantity and quality of apple yield and require control. The use of resistant apple cultivars may help to limit insecticide input and to increase the sustainability of pest management in apple orchards. Host plant resistance has a genetic basis, and increasing knowledge on the apple genome and QTL (quantitative trait locus) analysis helps to identify gene regions associated with resistance (Liebhard et al., 2003). QTL-based approaches to determine and characterize host-plant resistance against insects are commonly used in annual crops, however detailed analysis of the genetic basis of arthropod resistance in apple has received little attention (Bus et al., 2008; Stoeckli et al., 2008a). Contrary to diseases like scab or mildew, the variability in susceptibility to arthropod pests is generally low between the main apple cultivars. The detection of QTLs of minor effect is impeded by natural microsite-dependent variation of population density, additional to environmental parameters modifying or masking the expression of plant resistance. Within-tree variation in the distribution of leaf- (Unsicker & Mody, 2005) or fruitdamaging arthropods (Stoeckli et al., 2008b) has to be considered for sampling protocols aiming at quantifying the field resistance of particular tree genotypes. Effects of microsite- or management-dependent tree growth characteristics, and of the position of the studied 531
genotypes within an orchard in relation to other genotypes (neighbourhood effects) may also interfere with the characterization of the genetic basis of a genotype’s resistance. Water availability is an environmental parameter of paramount importance for plant growth and development. Temporary drought events are characteristic for many parts of the world, and frequency and intensity of extreme drought is predicted to increase in the future (Christensen et al., 2007). Water deficit may cause drought stress, which may have strong effects on plant resistance to arthropod herbivores. However, the influence of drought stress on plant resistance is not easily predictable, as both decreasing and increasing plant resistance as a consequence of drought stress has been observed (Huberty & Denno, 2004). Future studies, for example on stress intensity, are needed to better understand the effects of the environmental parameter drought stress on plant resistance (Mody et al., 2009). The goals of the presented studies were (1) to elucidate the postulated genetic basis of apple resistance to different species of arthropod herbivores by QTL analysis, and (2) to assess genotype-independent factors influencing the distribution of pest insects and plant resistance in the field and in the laboratory.
Material and methods QTL analysis and field experiments Resistance QTLs were investigated in the field in a segregating F1 cross of the apple varieties 'Fiesta' and 'Discovery' (Stoeckli et al., 2008a; 2009a,b). Progeny plants representing 160 genotypes were surveyed at three different sites in Switzerland (cantons Ticino, Valais and Zurich). Herbivore infestation per genotype as a measure of resistance in the field was quantified in two consecutive years for the rosy apple aphid (Dysaphis plantaginea), the leafcurling aphid (Dysaphis cf. devecta) and the green apple aphid (Aphis pomi), for the apple rust mite (Aculus schlechtendali), and for the codling moth (Cydia pomonella). QTL analyses based on herbivore infestation data were carried out with MapQTL® 4.0 (van Ooijen et al., 2002). The genetic linkage maps for both 'Fiesta' and 'Discovery' (single parent maps), used in QTL analysis, were calculated with 251 apple genotypes and were already published (Liebhard et al., 2003). Kruskal–Wallis tests and interval mapping (IM) were used for QTL analysis. Logarithm of odds (LOD) threshold values were determined by 1000-foldpermutation tests at a significance level of 95% (genome-wide). The same trees were also investigated to assess the possible importance of within-tree variation in herbivore distribution, of tree growth characteristics and of neighbourhood effects for the quantification of infestation by different apple pest arthropods. Within-tree variation of codling moth infestation was characterized based on a survey of 40’000 apples from 12 sectors of each of the 160 different apple genotypes, considering canopy aspect (north, east, south, and west) and canopy height (bottom, middle, and top) (Stoeckli et al., 2008b). Effects of shoot growth characteristics on aphid population development were repeatedly studied for population growth of the green apple aphid in sleeve cages attached to 200 apple trees of different genotype (Stoeckli et al., 2008c). Neighbourhood effects were assessed for apple aphids and rust mites by quantifying the relationship between infestation levels of neighbouring trees (Stoeckli et al., 2008a; 2009a). Laboratory experiments on the effects of drought stress on plant resistance Drought stress effects on apple resistance to a chewing and a sap-feeding herbivore (Spodoptera littoralis caterpillars; Aphis pomi aphids) were studied in laboratory experiments considering control conditions and two intensities of pulsed drought stress (Mody et al., 2009). 'Control' plants were maintained in constantly humid soil, 'low stress' plants were watered for the first time when leaves started drooping (about one week after start of the experiment), and 'high stress' plants before irreversible necrosis occurred. Herbivore
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experiments started after approximately three weeks of stress, i.e. 3 – 4 drought cycles for high stress plants and 6 – 7 drought cycles for low stress plants. Plants were watered during herbivore feeding to simulate different natural stress conditions with alternating dry and wet periods and insect feeding on plants that had previously been stressed, but were not while feeding actually occurred. As measures of resistance, plant acceptability (S. littoralis feeding preference in arena experiments; resistance by antixenosis) and plant suitability (S. littoralis growth rate and A. pomi population development; resistance by antibiosis) were quantified.
Results and discussion QTLs for herbivore resistance in apple We identified QTLs for herbivore resistance in apple. The detected QTLs highlight the genetic basis of arthropod resistance in apple. Apple genotypes amplifying QTL-relevant markers differed significantly from genotypes not amplifying the markers for the aphid species D. plantaginea and D. cf. devecta (Stoeckli et al., 2008a), the rust mite A. schlechtendali (Stoeckli et al., 2009a), and the codling moth C. pomonella (Stoeckli et al., 2009b) (Fig. 1). The detected markers may facilitate the breeding of resistant apple cultivars by marker assisted selection. They may also be used for screening existing cultivars for resistance to important pest arthropods. Environmental factors related to apple infestation by arthropod pests in the field In the field, additional environment-effects on herbivore distribution were identified. The infestation of apple fruits by the codling moth varied within apple tree canopies for first but not second generation larvae, with north-facing apples showing lower infestation than southor east-facing fruits (Stoeckli et al., 2008b). Population growth of the green apple aphid was positively related to the length and growth of apple shoots (Stoeckli et al., 2008c). Neighbourhood effects appeared to influence the infestation of apple trees by the rosy and the green apple aphids, but not by the leaf-curling aphid and the rust mite (Stoeckli et al., 2008a; Stoeckli et al., 2009a). These genotype-independent determinants of herbivore distribution may mask QTLs, and they may help to explain difficulties in QTL detection for the studied herbivore species.
Herbivore infestation (mean ± SE)
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Identified QTL marker present
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absent
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16 12 8
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4 0
D. plantaginea ! FiestaÕLG 17 E33M35-0269
D. cf. devecta ! FiestaÕLG 7 E32M39-0195
A. schlechtendali ! FiestaÕLG 7 E35M42-0146
C. pomonella ! DiscoveryÕLG 10 Z19-350
Figure 1. A significantly lower herbivore infestation was found for the subpopulation of the F1 apple cross amplifying the marker closest to the QTL compared to the apple genotypes not 533
amplifying the marker. Herbivore infestation was analyzed by Mann-Whitney U-test. Herbivore species, parent and linkage group (LG), closest marker to the QTL, and P-value (* P
