Characteristics associated with Woolly Apple ... - Wiley Online Library

Blackwell Publishing Ltd.

Characteristics associated with Woolly Apple Aphid Eriosoma lanigerum, resistance of three apple rootstocks W.R.M. Sandanayaka1, V.G.M. Bus2, P. Connolly1 & R. Newcomb1 The Horticulture and Food Research Institute of New Zealand Ltd; 1Mt Albert Research Centre, Private Bag 92169, Auckland, New Zealand; 2Hawkes Bay Research Centre, Private Bag 1401, Havelock North, New Zealand Accepted: 30 July 2003

Key words: Eriosoma lanigerum, apple rootstocks, resistant genes, phenology, Elecrical Penetration Graph, phloem feeding, Homoptera, Aphididae

Abstract

The resistance characteristics of the apple resistance genes (Er1, Er2, and Er3) to the woolly apple aphid, Eriosoma lanigerum (Hausmann) (Homoptera: Aphididae) were studied according to the performance measured on apple cultivars containing these resistance genes. The resistance characteristics of Northern Spy (Er1), Robusta 5 (Er2), and Aotea (Er3) were compared to the susceptible cultivar Royal Gala, by measuring the aphid settlement, development, and survival rates correlated with electronically monitored probing behaviour. Er1 and Er2 had a higher level of resistance with a significantly shorter period of phloem feeding, suggesting that the resistance factors were present in the phloem tissue. Phenological measurements indicated that the aphids showed poor settlement, development, and survival on Er2. Er1 also showed low settlement and survival, although not as low as Er2. Aphid performance and feeding on Aotea (Er3) were similar to Royal Gala, suggesting that some woolly apple aphids in New Zealand may have recently overcome Er3 resistance. The differences in resistance mechanisms of Er1, Er2, and Er3 are discussed in relation to the strategy of pyramiding these genes to give a durable resistance to woolly apple aphid.

Introduction The Woolly apple aphid (WAA), Eriosoma lanigerum (Hausmann) is an important pest of apples in many apple growing countries. It is most devastating on nursery trees and young trees in the orchard, but can also weaken mature trees by infesting both the stem and roots (Baker, 1915; Staniland, 1923; Monzen, 1925). Feeding activity of the aphid leads to the formation of galls on the roots and shoots (Staniland, 1924). Aphid colonies on the roots impact on both the number and weight of fruit (Brown et al., 1995). In many parts of the world, the direct impact above ground is seldom serious, as the aphid is controlled by a parasite, Aphelinus mali (Haldeman) (Hymenoptera: Aphelinidae), and predators such as the European earwig, Forficula auricularia L. (Dermaptera: Forficulidae), and the lady beetle, Parapriasus australasiae (Boisduval) (Coleoptera: *Correspondence: Insect Science Group, HortResearch, Mt Albert Research Centre, Private Bag 92169, Auckland, New Zealand. Email: [email protected]

Coccinellidae). Colonies on the roots, however, escape predation while they are also difficult to control by insecticides, therefore resistant rootstocks have and will continue to provide the preferred means of pest management. Three major WAA resistant genes have been identified and used in apple resistance breeding. They are Er1, Er2, and Er3, which are carried by the apple cultivars Northern Spy, Robusta 5, and Aotea, respectively. Northern Spy and its derivatives MM.106 and M.793 are commonly used resistant rootstocks in New Zealand. Resistance in Northern Spy is attributed to a dominant gene, Er1 (King et al., 1991). While WAA is known to sometimes feed on this cultivar, especially when tested under rigorous conditions in the glasshouse (Crane et al., 1936), it neither reproduces nor stimulates the formation of galls (Le Pelley, 1927; Massee, 1937; Roach, 1937). There have, however, been a few cases where new WAA biotypes have been reported to overcome the Er1 resistance (McClintock, 1930; Giliomee et al., 1968). These findings have instigated the quest for alternative sources of resistance in the apple germplasm (Rock & Zeiger, 1974; Cummins et al., 1981; Mackenzie & Cummins, 1982).

© 2003 The Netherlands Entomological Society Entomologia Experimentalis et Applicata 109: 63 – 72, 2003

63

64

Sandanayaka et al.

Robusta 5, carrying the Er2 gene (King et al., 1991), is immune to WAA. This cultivar has also been used extensively as a source of resistance in rootstock breeding programs (Cummins & Aldwinckle, 1974). In New Zealand, the evaluation of open-pollinated seedlings mainly derived from M. sieboldii, gave rise to the Aotea series of rootstocks (Taylor, 1981). They were selected for their resistance to WAA as well as other pests and diseases. One selection named ‘Aotea’ has been released, which was later shown to carry a major resistance gene, designated Er3 (Bus et al., 2000). The phenotyping of germplasm and breeding material is commonly based on a subjective measurement of the degree of infestation of the hosts following a certain period of time after inoculation. Some studies have included detailed observations on the survival, development, and colony establishment rates of the aphids to assess natural resistance in different cultivars, but still have not provided insight on the resistance mechanisms involved. However, studying the probing behaviour of insects on resistant and susceptible genotype in other plants has aided the localization of the resistance factors in host tissues (van Helden & Tjallingii, 1993). The Electrical Penetration Graph (EPG) measures the electrical signals during stylet penetration and feeding by aphids (Kimmins & Tjallingii, 1985; Spiller et al., 1985; Tjallingii, 1987; Tjallingii & Hogen Esch, 1993) and has proved an effective tool in studying resistance factors in aphid–plant relationships (Cole, 1994; Gabrys et al., 1997; Prado & Tjallingii, 1997; Ramirez & Niemeyer, 1999; Powell & Hardie, 2000). In this paper, we compare the characteristics associated with WAA resistance among three apple cultivars (Northern Spy, Robusta 5, and Aotea) to a susceptible cultivar (Royal Gala), by studying the phenological development and feeding behaviour of the aphid.

Materials and methods Plant material and insects

Trees of three resistant cultivars (Northern Spy, Robusta 5, and Aotea) and one susceptible cultivar (Royal Gala), all on resistant M.793 rootstocks were used in experiments. The trees were grown in 5 l plastic bags with potting mix in the nursery at the HortResearch Hawkes Bay Research Centre and pruned in such a way that they had one or two 1-month-old shoots for the 1st block of each experiment. Trees with shoots 2, 3, and 4 months old were tested in two, three, and four blocks, respectively. WAAs were collected from Hawkes Bay in 1999. A colony of aphids was established in a glasshouse (at 25 ± 5 °C) in Auckland Research Centre on a ‘Royal Gala’ apple tree from a single apterous virginoparae adult. Offspring

were used to infest more ‘Royal Gala’ and ‘Braeburn’ trees. Subsequent colonies were reared in a glasshouse at 25 ± 5 °C and in a cycle of 16 h light, 8 h dark (L16:D8), inside fine net cages to protect from parasites. Aphids reared on ‘Braeburn’ trees for three or four generations were used for the experiments to avoid adaptation of aphids to ‘Royal Gala’. Two different developmental stages of WAA were used in this study for the different experiments. The 1st instar nymphs were used for the aphid settlement, development, and survival observations on the trees, whereas early adult stages (1–2 days old) were used for Electrical Penetration Graph (EPG) tests. Phenological measurements

Apterous virginoparae adult aphids were separated from the colony and left in closed plastic square Petri dishes (9 × 9 × 2 cm) for 12 h to produce nymphs. Twenty-five nymphs were collected in a plastic vial (3 × 1.5 cm) with a 3 mm diameter hole at the bottom, plugged with cotton. Nymphs less than 24-h old were used in all experiments. The aphids were placed onto the longer shoot of the tree by attaching the closed plastic vial to a leaf node in the middle of the shoot with blu-tak® and the cotton plug was then removed to release the nymphs. A funnel shaped trap was made with laboratory film (Parafilm® ‘M’) at the base of the tree, 5 cm above the graft union, and filled with Vaseline® to trap the nymphs attempting to escape the tree. The trees of the four cultivars were then randomly positioned in the glasshouse. The experiments were carried out in a block design, one block per month, during the period November 2001–February 2002, in the glasshouse under reasonably controlled temperature (27 ± 5 °C). The 1st and 2nd blocks of the experiment were carried out in a L16:D8 light cycle in the glasshouse, and blocks 3 and 4 continued without glass house lights, due to the long daylight and increasing room temperature. The temperature was monitored every 15 min in the glasshouse, using a Tiny Tag™ measuring device and software. In each block, two experiments were carried out in parallel. In the first experiment, aphid settlement rates were measured on 10 trees per cultivar per block. Each tree was infested with one cage of 25 nymphs per tree on day 1 and monitored on days 4, 8, 12, 16, and 20, two trees per day. The number of aphids settled down on each plant was counted along with the numbers left in the plastic vials. The percentage settlement was calculated by taking the numbers settled on the plant, as a percentage of the total number minus the number remaining in the plastic vials. A magnifying lens (×4) was used for all observations on the trees. Developmental stages were established by counting the number of exuviae (indicating ecdysis) or offspring (indicating reproduction) immediately around each individual. Aphids surviving on each individual tree were removed, and

Woolly apple aphid resistance of three apple rootstocks 65

the body lengths from the head to the end of cauda were measured under a microscope. In the 2nd experiment in each block, the development rate and survival of the aphids from first instar to adult were measured. Two or three trees of each cultivar were inoculated with nymphs less than 24 h old using the method described above. To ensure that a satisfactory number of aphids would settle on the resistant cultivars, 50 nymphs were inoculated onto each of the trees. Observations were carried out daily, at the same time each day, using a magnifying lens (×4). The duration of each instar was determined by the presence of exuviae, and reproduction was recorded by the presence of offspring. Exuviae were carefully removed using a needle, without disturbing the aphids. Observations on development were continued until either the nymphs had moulted to the adult stages or disappeared from their original feeding site. The percentage survival was calculated by taking the total number of nymphs that completed the life cycle as a percentage of the total number of settling as first instar nymphs. In addition, qualitative measurements were taken on ‘wool’ or honeydew presence, colony establishment, and the formation of galls on the shoots. The differences between cultivars were analysed by using Generalized Linear Models (GLMs) in the statistical software R (Ihaka & Gentleman, 1996). A binomial model in which the number of settled aphids was weighted in proportion to the total number of aphids that had left the plastic vials was used for nymphal settlement, and a Poisson model was used for nymphal development periods. Body length measurements of the nymphs at different stages were analysed using a simple linear model. The models examined the effect of the blocks and instars, together with their interaction with the cultivar effects. EPG study of feeding behaviour

One- to two-day-old apterous virginoparae adult aphids were collected from the colony and placed on a clean Petri dish with a fine paintbrush. The aphids were checked under the microscope to ensure their mouthparts were not damaged. The white wax wool secreted from their bodies was removed with a moistened fine paintbrush. A droplet of conductive, solvent based silver paint (ProScitech, Australia) was deposited on the dorsum of the aphid and one end of a 2–3 cm long piece of gold wire (10 µm diameter) was attached to the silver paint droplet. The other end of the gold wire was attached to a copper wire. The wired aphids were placed on a Petri dish for 2 h to recover from the wire stress. (Preliminary tests on the time taken to attain the 1st phloem phase had been performed with wired aphids and free aphids to establish the length of the period required for the aphids to minimize wiring stress.) After the recovery

period, the wired aphid was connected to an EPG monitor (Giga-4, Wageningen Agricultural University, the Netherlands), which had 109 Ohm input resistance and an adjustable plant voltage. The aphid was then placed on a shoot with a diameter of 7–8 mm, with a limited freedom to move within the length of the gold wire. The plant electrode was inserted in the moistened soil mixture of the potted seedling and the circuit was completed when the aphid inserted its stylet into the plant. Input voltage was set at about 300 mV to establish an appropriate response of the signals. The electrical signals were acquired at 200 Hz through a Dataq A /D-converter card and Windaq lite data acquisition and analysis software on a PC computer. The waveforms recorded on the computer were saved as Windaq files and used later to determine the occurrence of different activities and the time spent for each activity in seconds. Recordings were made simultaneously on four plants, one aphid per plant. One tree of each cultivar was placed at random in the Faraday cage, at 23 ± 2 °C, under fluorescent light. New plants and aphids were used in each replicate and the probing behaviour of the aphids was recorded for 8 h. The experiments were repeated until at least 15 replicates per cultivar were obtained. When the aphids fell off the plants or tangled the wire during the recording period, the observations were excluded from the results. Readings were scored using the following parameters related to probing behaviour (Tjallingii, 1990): • np: total non-probing period during the experiment. • number of probes: total number of probes during the experiment. • 1st np: non-probing period of the first probe. • pathway phase: total duration of pp (preprobing phase: low voltage signal before the initial penetration), A waveform (initial penetration), B waveform (sheath salivation), C waveform (stylet pathway activities) and pd (potential drops corresponding to intracellular stylet tip punctures). • G: total duration of G waveform (xylem ingestion). • E1: total duration E1 waveform (salivery secretion into the phloem). • E1e: total duration of extracellular E1 waveform. • E1/(E1 + E2): E1 as a fraction of phloem phase (total duration of E1 and E2). • E2: total duration of E2 waveform (passive phloem ingestion). • E2 > 10 min: total duration of sustained phloem ingestion (continuous phloem ingestion more than 10 min). • E2/(E1 + E2): E2 as a fraction of phloem phase (total duration of E1 and E2). • E /exp.: time taken to attain the 1st phloem phase from the beginning of the experiment. • E /probe: time taken to attain the 1st phloem phase from the beginning of the first successful probe.

66

Sandanayaka et al.

Figure 1 Mean percentage settlement of woolly apple aphid on Royal Gala, Aotea, Northern Spy, and Robusta 5 for 4, 8, 12, 16, and 20 days following inoculation. Error bars show the standard errors.

The significant test for the parameters was carried out using the Kruskal–Wallis test. The different treatments were compared by Tukey-Kramer multiple comparison (α = 0.05) test (SAS Institute, 1995).

Results Settlement

The percentage settlement of woolly apple aphids varied among cultivars (Figure 1). In all four cultivars, the total number of nymphs trapped in Vaseline, the number that left in the plastic vial, and the number that settled on the tree were never equal to the number inoculated (25). It was assumed that the number of unaccounted nymphs had fallen off the trees without settling down. The binomial GLM used weighted the number settled and survived, in proportion to the total number of aphids that walked out

of the plastic vials. An analysis of deviance of the model used a χ2-test, which showed that all cultivars were significantly different from each other (P < 0.01). There was neither a block effect nor an interaction between block and cultivars. The highest level of percentage settlement and survival (33 ± 3.2) was recorded on Royal Gala and the lowest (1.6 ± 1.6) was on Robusta 5, including no settlement in blocks 2 and 3. The percentage settlement on Northern Spy was lower than Royal Gala but higher than Aotea. A slight decrease of percentage settlement was noticeable over time in Northern Spy (Figure 1). However, including time in the model showed no interaction between time and variety (χ2 = 1.19, d.f. = 3, P < 0.59) and time itself had a minimal effect (χ2 = 3.16, d.f. = 1, P < 0.08). The survival rate from the 1st instar nymph to adult was 100% on Royal Gala and Aotea, but 45% on Northern Spy and none on Robusta 5 (Table 1). It was noted that the survival of the nymphs on

Table 1 Development period and survival of nymphal stages (mean ± SE) of E. lanigerum reared on Royal Gala, Aotea, Northern Spy, and Robusta 5 Mean nymphal period (days) Cultivar

Instar 1

Instar 2

Instar 3

Instar 4

Total

Royal Gala

5.36 ± 0.20 (n = 66) 6.14 ± 0.16 (n = 56) 7.16 ± 0.46 (n = 38) 6.00 ± 1.02 (n = 8)

2.83 ± 0.11 (n = 66) 2.75 ± 0.11 (n = 56) 4.29 ± 0.31 (n = 34 3.57 ± 0.43 (n = 7) )

2.55 ± 0.10 (n = 66) 2.75 ± 0.10 (n = 56) 3.67 ± 0.39 (n = 21) 4.00 (n = 1)

2.47 ± 0.10 (n = 66) 2.66 ± 0.09 (n = 56) 4.29 ± 0.29 (n = 17) –

13.21 ± 0.38 (n = 66) 14.30 ± 0.27 (n = 56) 16.65 ± 0.74 (n = 17) –

Aotea Northern Spy Robusta 5

*Percentage survival from 1st instar to adult.

Percentage survival* 100 100 45 0


Northern Spy decreased with time. On Royal Gala and Aotea, aphids stayed mostly at one feeding site through the nymphal stages to the adult stage and further. In contrast, aphids were often observed to move on Northern Spy, particularly after ecdysis. During our observations, only the exuviae were found at the places where the aphids had originally settled, and new settlements were found towards the tender part of the shoot. On Northern Spy, some of the nymphs disappeared after the 1st, 2nd, or 3rd instar and the nymphs that moved from their original feeding site could not complete their lifecycle. Development

The rate of body length increase in WAA varied among cultivars. Robusta 5 was not considered in this comparison as it had only a total of seven data points. The body lengths were taken to measure the growth of the nymphs on different plant types, referring to the findings of Asante (1994), that there is a positive correlation between fresh body weights and body lengths of adult apterous virginoparae WAAs. Royal Gala had the highest rate of body length development and Northern Spy the lowest (Figure 2). The shortest nymphal period was recorded on Royal Gala (13.21 ± 0.38 days) and the longest on Northern Spy (16.65 ± 0.74 days). While recording data daily, we observed feeding difficulties of WAA on Northern Spy. Nymphs that moved to a few different places mostly towards the younger part of the shoots, could not develop into adults. The nymphs that became adults and started to reproduce on Northern Spy, stayed at their original feeding site but the offspring moved to the

tender part of the shoots. A marked interaction between block and cultivar is evident from the body length measurements taken on days 8, 12, and 16 (Figure 2). However, with the measurements taken on day 20, when the slow-developing nymphs developed into later instars or adults, the interaction between block and cultivar disappeared with a much smaller effect of block still evident. By day 20, some of the aphids on Northern Spy were still in their 3rd or 4th instars, but all the other aphids were adults. These findings were further confirmed in experiment 2. The development period from the 1st instar nymph to the adult varied among cultivars. The development rate of all instars was higher in later blocks than earlier blocks, with none of the aphids developing into adults on Northern Spy in the last block in February 2002. A Poisson GLM was used to analyse the duration of nymphal development from 1st instar to adult, with a χ2test used in the analysis of deviance associated with the GLMs to ascertain statistical differences. The nymphs on Robusta 5 did not remain after the 2nd instar. Among the other three varieties, the aphids on Northern Spy took longer to develop than on the other two varieties. The duration of 1st instar to adult was the same on Royal Gala and Aotea, but Royal Gala was always different from Northern Spy. Aotea was different from Northern Spy in Block 2 (χ2 = 5.0975, P < 0.025) and Block 3 (χ2 = 5.2928, P < 0.025), but not in Block 1 (χ2 = 2.3794, P > 0.1). Block 4 stands out, in that none of the aphids survived after the 3rd instar on Northern Spy, with the cultivar effect being most noticeable in Block 3.

Figure 2 Box plots of body lengths of woolly apple aphids developed on Royal Gala, Aotea, and Northern Spy after 4, 8, 12, 16, and 20 days since inoculation. Black dot: median; vertical lines: maximum and minimum values; open rectangle: between 1st and 3rd quartile; circle: outlier.

68

Sandanayaka et al.

Wool production

Gall formation

We observed similar amounts of ‘wool’ being produced by aphids on Royal Gala and Aotea. Wool production on Northern Spy or Robusta 5 was always less than on Royal Gala and Aotea. Aphids on Northern Spy and Robusta 5 did not produce wool in their early stages.

On Royal Gala and Aotea, galls became visible as swellings of the bark, at the feeding sites of the individuals, as they reached the 3rd or 4th instar. This swelling gradually increased with the growth of the aphid. When the offspring emerged, they settled down on the gall, forming a colony. On Northern Spy, the galls were not very obvious, but could be detected by touch on the bark. Although a few aphids completed their lifecycle on Northern Spy, none of them made colonies. No galls were observed on Robusta 5.

Honeydew production

Honeydew produced by individual aphids was used as evidence of feeding. The most honeydew was produced on Royal Gala. Aphids on Aotea produced either equal or less honeydew than Royal Gala but more than on Northern Spy. Aphids on Robusta 5 produced hardly any honeydew. Colony establishment

Adult aphids established colonies at their feeding sites on Royal Gala and Aotea. Colony establishment was not observed on Northern Spy, as the offspring dispersed to the younger parts of the trees and the adults often disappeared from their original reproductive site. No colony establishment was observed on Robusta 5, as none of the aphids survived until the reproductive stage.

Feeding behaviour

The results of the comparison of EPG parameters among four different cultivars are summarized in Table 2. Since no detailed discussion of the EPG characteristics of WAA has been undertaken elsewhere, we have used a new parameter (pp), to the signal pattern, which appeared before the A waveform (Sandanayaka & Hale, 2003). The B and C waveforms often overlapped and the pp, A, B, and C waveforms with numerous potential drops were pooled as pathway phase to avoid losing any information. Xylem ingestion (waveform G), with a frequency varying from 6 to 11 Hz, was common in all cultivars. The E1 waveform in extracellular

Table 2 Mean values (mean ± SE) of EPG parameters in Royal Gala, Aotea, Northern Spy, and Robusta 5 for an 8 h period. Duration in rows 1, 3, 4, 5, 6, 7, 9, 10, 12, and 13 are given in minutes EPG parameters

Royal Gala (n = 15)

Aotea (n = 15)

Northern Spy (n = 15)

Robusta 5 (n = 17)

1 2 3 4 5 6 7

157.20 ± 28.46 2.47 ± 0.45 30.48 ± 9.43 121.03 ± 17.11 78.12 ± 9.00 47.98 ± 10.93 7.16 ± 2.40

159.36 ± 27.82 2.20 ± 0.31 47.38 ± 9.36 153.25 ± 22.49 61.57 ± 16.02 21.68 ± 6.53 6.79 ± 5.11

242.43 ± 34.00 2.40 ± 0.47 45.48 ± 12.47 123.11 ± 19.83 80.60 ± 18.40 22.86 ± 14.64 7.95 ± 7.39

257.38 ± 38.57 1.53 ± 0.23 57.78 ± 17.37 95.27 ± 19.59 58.52 ± 19.38 57.57 ± 26.07 52.51 ± 26.34

0.45 ± 0.07a

0.35 ± 0.08a

75.98 ± 17.69a 49.33 ± 14.05ab

84.44 ± 26.74a 80.66 ± 26.54b

0.55 ± 0.07a

0.65 ± 0.08a

179.48 ± 28.75 79.14 ± 18.02 100 73.3

116.05 ± 17.98 66.45 ± 12.91 73.3 60

np number of probes 1st np pathway phase G E1 E1e E1 8 E1 + E 2 9 E2 10 E2 > 10 min E2 11 E1 + E 2 12 E/exp. 13 E/probe % of aphids attained phloem % of aphids showed E2 > 10 min

0.55 ± 0.13ab 11.30 ± 5.7b 6.83 ± 4.73a 0.45 ± 0.13ab 219.08 ± 35.58 99.50 ± 28.37 53.3 20

0.80 ± 0.08b 11.6 ± 5.72b 8.00 ± 4.53a 0.20 ± 0.08b 114.04 ± 15.73 65.52 ± 17.16 41.2 23.5

Means followed by different letters within rows (8, 9, 10, and 11) are significantly different according to the Tukey–Kramer multiple comparison test (∝ = 0.05) (SAS Institute, 1995). np: total non-probing period, C: total duration of pp, A, B, C waveforms including potential drops, G: total duration of xylem ingestion, E1: total duration of salivery secretion into phloem, E2: passive phloem ingestion, E/exp. time to 1st phloem phase from the beginning of the experiment, E/probe: time to 1st phloem phase from the beginning of the successful probe.


voltage level (E1e) occurred frequently in spite of the cultivar difference. Since the biological correlation of E1e is unknown (van Helden & Tjallingii, 2000), the phloem salivation, which was characterized by E1, appeared in intracellular voltage level. A comparison of the 13 EPG parameters across cultivars was carried out using both non-parametric (Kruskal–Wallis rank sums test) and parametric (Tukey–Kramer multiple comparison test) tests. Both tests gave similar results in all cases, except E1 and E1e, which were found to be significant at P < 0.05 using the Kruskal–Wallis test but not significant using the Tukey–Kramer test. Since the parametric tests are more powerful, we drew our conclusions based on Tukey– Kramer multiple comparison test. The total duration of individual events of EPG signal patterns; np, 1st np, pathway phase, G, E1, E1e, and number of probes were not significantly different among cultivars. E /exp. and E /probe were also not significantly different among cultivars. E1 as a fraction of E1 + E2 of Robusta 5 was significantly higher than Royal Gala and Aotea, but not different from Northern Spy, which indicates more salivation during the phloem phase (total E1 and E2) on Robusta 5. E1 /(E1 + E2) in Northern Spy is not significantly different from the other three cultivars. The most striking differences between the four cultivars tested was in E2, where the aphids spent longer in the phloem ingestion on Royal Gala and Aotea than Northern Spy and Robusta 5. The mean duration of sustained phloem ingestion (E2 > 10 min) by aphids on Northern Spy and Robusta 5 was significantly lower than on Aotea. The E2 as a fraction of E1 + E2 in Robusta 5 was significantly lower than Aotea and Royal Gala, but not different from Northern Spy, which indicates less passive ingestion during phloem phase. One hundred per cent of aphids on Royal Gala reached the phloem phase, and 73.3% showed sustained phloem ingestion. On Northern Spy and Robusta 5, only 53.3% and 41.2% of aphids reached the phloem phase, and only 20% and 23.5% of the aphids exhibited sustained phloem ingestion, respectively.

Discussion Plant-based resistance is a preferred option for managing pests on crops (Crane et al., 1936; Cole, 1994; van Helden & Tjallingii, 2000). However pests can overcome such resistance. This has created the need to produce durable plant-based resistance by pyramiding resistance genes. If the resistances being pyramided are conferred by independent mechanisms, it is likely that such mechanisms would require multiple evolutionary changes in the aphid to overcome them. WAA resistant cultivars of apple have been extensively used in apple rootstock breeding programs. While each of the three resistant cultivars have been found to contain different genes

conferring resistance (Er 1, 2, and 3), the question remains whether a unique resistance mechanism is conferred by each gene. The behavioral physiology of WAA on resistant cultivars was compared with reference to the commercial WAA susceptible cultivar Royal Gala. Royal Gala displays a range of WAA susceptible characteristics, including a high level of settlement, survival, rapid development, and long periods of phloem ingestion. One hundred per cent of the nymphs that settled on Royal Gala developed into adults within a 9–19 day period. This is consistent with other studies on developmental time of WAA (Monzen, 1925; Mackenzie & Cummins, 1982; Asante & Danthanarayana, 1990). We found that development time for the first instar was longer than the remaining three instars in each individual aphid on Royal Gala, as well as other cultivars, confirming the findings of Asante et al. (1991). When compared to Royal Gala, Northern Spy (Er1) showed resistance characteristics, including low levels of settlement and survival, slow developmental rate, long development period, and short phloem ingestion. Although 53% of the adults reached the phloem phase, and 20% of them had sustained phloem ingestion on Northern Spy, the comparative total duration of phloem feeding (E2) was significantly lower than Royal Gala. The reduced phloem sap ingestion was often the main feature associated with resistance, as was reported in EPG studies performed on other aphid/plant combinations (Campbell et al., 1982; Dorschner & Baird, 1989; Mesfin et al., 1992; van Helden & Tjallingii, 1993; Cole, 1994; Paul et al., 1996; Sauge et al., 1998). Accordingly, we suggest that the active resistance factors in Northern Spy may exist in the phloem, due to the significantly lower total duration of the phloem ingestion. Nymphs tended to move towards the tender parts of the shoots and there was a lower survival in later blocks of our experiment. Taken together, these observations suggest that resistance levels may increase with the maturity of the tree. Gall formation is an important feature in colony establishment. Although we have not seen the establishment of colonies on Northern Spy, small galls could be observed where the aphids stayed longer on the trees. This agrees with Bramstedt (1938), who reported that resistant varieties form some gall tissues and necrotic lesions, but the cells of gall tissue remain small compared to those of the susceptible varieties. In contrast, Staniland (1923) reported that the aphids settled down on Northern Spy for a very short time, but cannot feed for long and never formed galls. The results of the present study agree with the findings of Le Pelley (1927), who observed that an individual aphid could live on the shoots of Northern Spy with its stylet inserted for more than 5 weeks. Knight et al. (1962) reported that Northern Spy resistance has not broken down to full susceptibility and that aphids were not

70

Sandanayaka et al.

capable of colonising on the trees. Underhill & Cox (1938) found that Northern Spy, when grown on their roots, displayed definite resistance to WAA establishment and formation of galls on the roots. The decreasing survival of nymphs with their growth on Northern Spy (Table 1) confirmed the findings of Mackenzie & Cummins (1982), who reported that the survival on Northern Spy was 43% after 1 week, 30% after 2 weeks, 8% after 3 weeks, and 6% after 4 weeks. The significantly shorter duration of phloem ingestion on Robusta 5 may indicate that a factor in the phloem is the cause of the poor survival of the nymphs, lack of colony establishment, and failure to initiate gall formation on this cultivar. None of the nymphs completed their lifecycles on Robusta 5, and a few survived up to the 2nd or 3rd instar. 41.2% of adults monitored on EPG, reached the phloem phase, with 23.5% showing the sustained phloem ingestion. This confirms the findings of Crane et al. (1936), who reported that, even immune varieties occasionally appear susceptible to attack under glasshouse conditions. Cummins & Aldwinckle (1974) also reported that Robusta 5 was highly resistant or immune to WAA. Colonization of the WAA could not be achieved on Robusta 5 after repeated inoculations (Cummins et al., 1981) and nymphal survival was low. For example, Mackenzie & Cummins (1982) recorded 2% nymphal survival on Robusta 5 after 1 week. Adult aphids on Aotea (Er3) had significantly longer phloem ingestion periods than aphids feeding on the other two resistant cultivars. In addition, 73% of the adults monitored on Aotea reached the phloem phase and 60% showed sustained phloem ingestion. These values are approximately threefold higher than on the other resistant cultivars. The phenological and feeding characters of aphids on Aotea did not differ significantly from those on Royal Gala, except for a poorer settlement following artificial inoculation. However, higher levels of settlement were observed in the second generation on the trees as the colonies were established around the adults’ feeding site, which by then induced the formation of galls for the offspring to feed on. The gall formation, wool production, and colony establishment on Aotea were also not different from Royal Gala. In contrast, the Er3 resistance has been very stable in the field, where no aphids were found to settle on the resistant progeny of a cross between M.9 (a susceptible rootstock cultivar) and Aotea until at least 1998 (Bus et al., 2000). However, 4 years later in 2002, WAA settled on nine progeny following the re-inoculation of a subsample of 10 resistant progeny from this M.9 × Aotea cross in the greenhouse with aphids collected in the HortResearch orchard in Hawkes Bay. This finding strongly supports the suggestion that WAA has adapted to overcome Er3- mediated resistance. A better understanding of the mechanisms of resistance may give some

insight to the question of why the Er3 gene in Aotea was overcome by WAA. That WAA has developed virulence to Er3 but not to Er1 and Er2, suggests that Er3 mediated resistance is distinct from Er1 and Er2. Er1 and Er2 both had phloem associated resistance. The failure to detect significant differences in time from the beginning of the experiment to the 1st phloem phase (E /exp.) and in time from the beginning of a successful probe to the 1st phloem phase (E/probe) is consistent with a phloem-based resistance mechanism. The difference in response across blocks suggests that Er1 and Er2 may show distinct temporal patterns of resistance expression. Expression of resistance conditioned by Er1 appeared to increase with tree maturity, while resistance conditioned by Er2 was high from the earliest stages of tree maturity tested. Additional experiments are needed to confirm this trend. If subsequent research confirms the existence of two independent mechanisms, pyramiding Er1 and Er2 may enhance the durability of both genes. While some WAA have overcome the Er3 resistance gene, it is possible that the resistance factor still confers a partial level of resistance to the aphid. Therefore, pyramiding this broken resistance factor with Er1 and Er2 may further increase the durability of the two genes.

Acknowledgements We are grateful to Dr W.F. Tjallingii for advice on identification and analysis of EPG signal patterns. We further thank Drs Kim Plummer and Erik Rikkerink for comments on the manuscript and Dr Nihal De Silva for help with the statistics. This work was funded by the New Zealand Foundation for Research, Science and Technology.

References Asante SK & Danthanarayana W (1990) Laboratory rearing of the woolly aphid, Eriosoma lanigerum (Hausmann) (Hemiptera: Aphididae). Plant Protection Quarterly 5: 52–54. Asante SK, Danthanarayana W & Heatwole H (1991) Bionomics and population growth statistics of apterous virginoparae of woolly apple aphid, Eriosoma lanigerum, at constant temperatures. Entomologia Experimentalis et Applicata 60: 261–270. Asante SK (1994) Seasonal occurence, development and reproductive biology of the different morphs of Eriosoma lanigerum (Hausman) (Hemiptera: Aphididae) in the northern tablelands of New South Wales. Journal of Australian Entomological Society 33: 337–344. Baker AC (1915) The Woolly Apple Aphis. pp. 1–55. United States Department of Agriculture, Washington DC. Bramstedt F (1938) A histological method of determining the immunity of apple varieties to woolly aphis. Zeitschrift für Pflanzenkrankheiten und Pflanzenschutz 48: 480 – 488.


Brown MW, Schmitt JJ, Ranger S & Hogmire HW (1995) Yield reduction in apple by edaphic WAA (Homoptera: Aphididae) populations. Journal of Economic Entomology 88: 127–133. Bus V, Ranatunga C, Gardiner S, Bassett H & Rikkerink E (2000) Marker assisted selection for pest and disease resistance in the New Zealand apple breeding programme. Acta Horticulturae 538: 541–547. Campbell BC, McLean DL, Kinsey MG, Jones KC & Dreyer DL (1982) Probing behavior of the greenbug (Schizaphis graminum, Biotype C) on resistant and susceptible varieties of sorghum. Entomologia Experimentalis et Applicata 33: 140 –146. Cole RA (1994) Locating a resistance mechanism to the cabbage aphid in two wild Brassicas. Entomologia Experimentalis et Applicata 71: 23 –31. Crane MB, Greenslade RM, Massee AM & Tydeman HM (1936) Studies on the resistance and immunity of apples to the woolly apple aphis, Eriosoma lanigerum (Hausm.). Journal of Pomology 14: 137–163. Cummins JN & Aldwinckle HS (1974) Breeding apple rootstocks. Hortscience 9: 367–372. Cummins JN, Forsline PL & &Mackenzie JD (1981) Woolly apple aphid colonization on Malus cultivars. Journal of American Society of Horticultural Science 106: 26 –30. Dorschner KW & Baird CR (1989) Elecronically monitored feeding behavior of Phorodon humuli (Homoptera: Aphididae) on resistant and susceptible hop genotypes. Journal of Insect Behavior 2: 437– 447. Gabrys B, Tjallingii WF & van Beek TA (1997) Analysis of EPG recorded probing by cabbage aphid on host plant parts with different glucosinolate contents. Journal of Chemical Ecology 23: 1661–1673. Giliomee JH, Strydom DK & van Zyl HJ (1968) Northern Spy, Merton and Malling-Merton rootstocks susceptible to woolly aphid, Eriosoma lanigerum, in the Western Cape. South African Journal of Agricultural Science 11: 183 –186. van Helden M & Tjallingii WF (1993) Tissue location of lettuce resistance to the aphid Nasonovia ribisnigri using elecrical penetration graphs. Entomologia Experimentalis et Applicata 68: 269 –278. van Helden M & Tjallingii WF (2000) Use of electrical penetration graphs in plant resistance research. Principals and Applications of Electronic Monitoring and Other Techniques in the Study of Homopteran Feeding Behaviour (ed. by GP Walker, EA Backus), pp. 144 –172. Thomas Say Publications in Entomomlogy, USA. Ihaka R & Gentleman R (1996) R: a language for data analysis and graphics. Journal of Computational and Graphical Statistics 5: 299 –293. Kimmins FM & Tjallingii WF (1985) Ultrastructure of sieve element penetration by aphid stylets during electrical recording. Entomologia Experimentalis et Applicata 39: 135 –141. King GJ, Alston FH, Battle I, Chevreau E, Gessler C, Janse J, Lindhout P, Manganaris AG, Sansavini S, Schmidt H & Tobutt K (1991) The ‘European Apple Genome Mapping Project’ – developing a strategy for mapping genes coding for agronomic characters in tree species. Euphytica 56: 89–94. Knight RL, Briggs JB, Massee AM & Tydeman HM (1962) The inheritance of resistance to woolly aphid, Eriosoma lanigerum

(Hsmnn.) in the apple. Journal of Horticultural Science 37: 207–218. Le Pelley R (1927) Studies on the resistance of apple to the woolly aphis (Eriosoma lanigerum Hausm.). Journal of Pomology and Horticultural Science 6: 209 –241. Mackenzie JD & Cummins JN (1982) Differentiation of Malus clones into resistance classes by their effects on the biology of Eriosoma lanigerum Hausmn. Journal of American Society of Horticultural Science 107: 737–740. Massee AM (1937) Entomological Technique – Proceedings of the Association of Applied Biologists. Annals of Applied Biology 24: 195 –198. McClintock JA (1930) The source of apple seed in relation to pests on seedlings. Proceedings of American Society of Horticultural Science 27: 120 –123. Mesfin T, Thottappilly G & Singh SR (1992) Feeding behaviour of Aphis craccivora (Koch) on cowpea cultivars with different levels of aphid resistance. Annals of Applied Biology 121: 493–501. Monzen K (1925) The woolly aphid (Eriosoma lanigera Hausm.) in Japan, with special reference to its life history and the susceptibility of the host plant. Entomologen-Kongreβ Verh. 111: 249 –279. Paul TA, Darby P, Green CP, Hodgson CJ & Rossiter JT (1996) Electrical penetration graphs of the damson-hop aphid, Phorodon humuli on resistant and susceptible hops (Humulus lupulus). Entomologia Experimentalis et Applicata 80: 335 – 342. Powell G & Hardie J (2000) Host-selection behaviour by genetically identical aphids with different plant preferences. Physiological Entomology 25: 54 –62. Prado E & Tjallingii WF (1997) Effects of previous plant infestation on sieve element acceptance by two aphids. Entomologia Experimentalis et Applicata 82: 189 –200. Ramirez CC & Niemeyer HM (1999) Salivation into sieve elements in relation to plant chemistry: the case of the aphid Sitobion fragariae and the wheat, Triticum aestivum. Entomologia Experimentalis et Applicata 91: 111–114. Roach WA (1937) Studies on possible causes of immunity – Proceedings of the Association of Applied Biologists. Annals of Applied Biology 24: 206 –210. Rock GC & Zeiger DC (1974) WAA infests Malling-Merton rootstocks in propagation beds in North Carolina. Journal of Economic Entomology 67: 137–138. Sandanayaka WRM & Hale CN (2003) Electronically monitored stylet pathway of woolly apple aphid, Eriosoma lanigerum (Hausmann) (Homoptera: Aphididae). New Zealand Journal of Crop and Horticultural Science 31: 107–113. SAS Institute (1995) JMP user’s guide, version 3.1. SAS Institute, Cary, NC, USA. Sauge MH, Kervlla J & Rahbé Y (1998) Probing behaviour of the green peach aphid Myzus persicae on resistant Prunus genotypes. Entomologia Experimentalis et Applicata 89: 223 –232. Spiller NJ, Kimmins FM & Llewellyn M (1985) Fine structure of aphid stylet pathway and its use in host plant resistance studies. Entomologia Experimentalis et Applicata 38: 293– 295.

72

Sandanayaka et al.

Staniland LN (1923) The immunity of apple stocks from attacks of woolly aphis (Eriosoma lanigerum, Hausmann), part I. The relative resistance of different root stocks. Journal of Pomology and Horticultural Science 3: 85 –95. Staniland LN (1924) The immunity of apple stocks from attacks of woolly aphis (Eriosoma lanigerum, Hausmann), part II. The causes of the relative resistance of the stocks. Bulletin of Entomological Research 15: 157–170. Taylor JB (1981) The selection of Aotea apple rootstocks for resistance to woolly aphis and to root canker, a decline and replant disease caused by basidiomycete fungi. New Zealand Journal of Agricultural Research 24: 373 – 377. Tjallingii WF (1987) Stylet penetration activities by aphids: new

correlations with electrical penetration graphs. Insects Plants (ed. by V Labeyrie, G Fabres & D Lachaise), pp. 301–306. Dr W. Junk publishers, Netherlands. Tjallingii WF (1990) Stylet penetration parameters from aphids in relation to host-plant resistance. Proceedings VIIth International Symposium on Insect Plant Interactions. Symposia Biologica Hungarica 39: 411– 419. Tjallingii WF & Hogen Esch Th (1993) Fine structure of aphid stylet routes in plant tissues in correlation with EPG signals. Physiological Entomology 18: 317–328. Underhill GW & Cox JA (1938) Studies on the resistance of apple to the WAA, Eriosoma lanigerum (Hausm.). Journal of Economic Entomology 31: 622– 625.