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The overall objective was to employ genomic techniques to probe gene expression changes as the RAA interacts with its hosts, in order to expose physiological stresses on the pest and determine if any of these stresses are potentially exploitable in advancing control strategies.

To achieve this the specific objectives were to:

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  Determine changes in gene expression profile and characterise genes involved during the aphid’s period on apple leading up to host alternation and then surviving on its secondary host
  
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  Determine differences in gene expression profile and characterise genes involved when RAA is reared on apple cultivars of high and low susceptibility
  
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  Interpret the physiological underpinning to differential gene expression during host alternation and on alternative cultivar hosts, as a potential means to identifying future exploitable control possibilities
  

Information was sought from the literature and from discussions with researchers who have worked on apple (EMR apple breeders and entomologists, and the curator at the National Fruit Collection) to produce a list of candidate cultivars of diverse origin that may hold some degree of tolerance/resistance to RAA (Annex 1). Most candidates came from reports based on observations of natural infestation levels in orchards (Briggs and Alston,1969; Haltrich et al., 2000; Cross and Berrie, 2000, 2002, 2003, 2004; Andreev and Kutinkova, 2004; Cross et al., 2005; Arnaoudov and Kutinkova, 2006). The difficulty with these is the often low incidence and patchy distribution of RAA in orchards, which means that it is often not possible to make firm conclusions about a cultivar’s susceptibility to RAA. A cultivar may be free of RAA wholly through random chance. It was therefore likely that a high number of candidates might prove negative for resistance and so a high number, 26, were chosen for analysis. Some candidates did, however, come from artificial inoculation studies (Lyth, 1985; Rat-Morris, 1993; Mianarro and Dapena, 2004; Ngeli and Simoni, 2006) and from books on apples (Morgan and Richards, 2002).

Graftwood was collected from the 26 candidate cultivars from the EMR apple collection and from the Brogdale National Fruit Collection of over 2,300 different varieties of apple, to produce testable potted plants. Scions were bench-grafted onto M26 semidwarfing rootstocks in pots of pesticide free compost and placed in a greenhouse for 3 months to take. After staking, plants were moved to a gauzehouse ready for experiments. Various measures were taken to keep the plants pest free. If mildew became a problem plants were either removed or sprayed with Nimrod. Unwelcome aphids (Aphis pomi and Macrosiphum insertum) were killed by hand, or if they became unmanageable they were sprayed with the short-lived aphicide, Aphox, away from the gauzehouse.

Every winter the plants were cut back to within three buds of the graft. This produced fresh shoots in spring and kept the plants a manageable size within the gauzehouse.

4. APPLE INOCULATION AND APHID SAMPLING EXPERIMENTS

4.1 Qualitative apple susceptibility screens

At the start of the project the first inoculation experiments needed to uncover resistant cultivars relatively quickly and provide enough aphids from them to harvest for the genomics work. This has proved crucial because the resistances of some cultivars was found to be particularly strong, with very few aphids surviving very long and therefore requiring constant replacement and harvesting.

More quantitative experiments were conducted in a separate gauzehouse in 2006 and 2007 by an entomologist, and are reported in section 4.3.

4.1.1 2005 inoculations

In 2005, inoculations were done on three blocks of 12 cultivars. Three of these cultivars are widely reported as being highly susceptible to RAA attack (Bramley, Discovery, and Greensleeves). The nine other cultivars had at some time been referenced in the literature as possibly having some uncharacterised tolerance/resistance towards RAA (Saturn, McIntosh, Ontario, Liberty, Florina, Malus robusta 3760, Malus floribunda 821, Priscilla, Goldrush).

Each block was inoculated with a different RAA clone collected from three infestations in the field early in the season. Clones were used so the genetic background of each aphid was identical, and the three clones were taken from different cultivars (Red Falstaff, Discovery, Florina) to eliminate any preadaptation effects.

Plants were inoculated at the 7-8 leaf stage. At inoculation, each plant had four mature aphids placed on the upper surface of the uppermost leaf, and left to establish for 2 weeks. Where necessary plants were reinoculated until four aphids were seen to have settled. Four adult aphids were then harvested every week (for 5 weeks) from each plant, frozen immediately in liquid nitrogen, and stored at -80°C until RNA extraction. For plants that produced no growing colonies, fresh adult aphids were introduced.

In 2006 and 2007, the same 12 cultivars as 2005 were rescreened and sampled this time as four replicate lines rather than blocks, to aid aphid harvesting.

As is reported in the next section, the resistances of some cultivars was very strong, with very few aphids surviving very long and therefore requiring constant replacement, in order to get enough aphids to harvest. So this time, at inoculation, each plant had ten mature aphids placed on its uppermost leaf. As most aphids eventually walked off some ‘resistant’ cultivars, a Vaseline band was put around the base of the main stem and/or clip cages were employed to force the aphids to feed on the apple (and so express genes in response to that cultivar’s resistance mechanism). Eight adult aphids were harvested from each plant when possible, frozen immediately in liquid nitrogen, and stored at -80°C until RNA extraction. Only aphids that were physically seen to be feeding on the plants were sampled.

In 2006, an additional eight cultivars were screened (Northern Spy, Wagener, Cox, Fiesta, St Lawrence, Fameuse, Hoary Morning, Worcester Pearmain) and, in 2007, an additional five (Belgolden NO 17, Mollie’s Delicious, Aivaniya, Double Red Wealthy, Delorina, Malus tschonoskii).

At inoculation, the settling behaviour was observed for any antixenotic (deterrent) effects. Without exception all the adults moved to the underside of the leaf and immediately began feeding and invariably then laid a nymph.

Every day the adults were observed for numbers, activity (feeding or walking), location of aphids feeding (apex, leaves, stems) and presence of winged morphs. The seedlings were scored for leaf curl.

Table 1 below summarises the progression of infestation following inoculations in early June.

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  Six of the cultivars (Bramley, Discovery, Florina, Greensleeves, McIntosh, Saturn) are clearly highly susceptible with the plants developing heavy infestations by the first score date in mid-June, resulting in extensive leaf curl. They also produced wing morphs within the first or second score date.
  

The remaining six showed varying levels of resistance.

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  Priscilla produced very small aphids and slow colony growth localised on the very top growing leaves, which produced winged aphids only at the forth score date. No leaf curl was seen
  

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  Goldrush, Liberty and M. floribunda all produced very small aphids and slow colony growth localised on the stem until the latest score date, when they moved onto the leaves to produce a few winged aphids. No leaf curl
  

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  M. robusta was highly resistant but reacted to the aphids with shoot tip death
  

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  Ontario was highly resistant with aphids constantly on the move and not feeding. No winged aphids were produced and no leaf curl was produced
  

The results of this screen are as follows: