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Table 3. Nymphal production and mortality for different apple cultivars which had been inoculated with single adult aphids at day 0 (total of seven replicates)


Apple cultivar

Day 1




Day 4




Total live nymphs

Total dead nymphs

Total live nymphs

Total dead nymphs

Ontario

28

0

10

14

Bramley

53

0

262

1

Greensleeves

56

0

204

1

Liberty

55

0

199

2

Ontario clearly leads to substantial nymph death. Liberty’s resistance is not evident in this experiment in terms of aphid mortality.


4.3.3 Autumn returns to apple cultivars
Methods
Three cvs, Bramley, Priscilla and Ontario, were assessed for their preference to autumn gynoparae. Pot grown apple seedlings were placed in a gauzehouse, in an open circle (to allow access), in a randomised block design. The three different cultivars were classed as treatments and there were five replicates/blocks. Two hundred RAA gynoparae cultured in the laboratory on plantain in short day lengths were released from a flexiglass container in the centre of the trees on 8 November 2007. The number of gynoparae on each tree was assessed after 24 h. Leaf colour was also measured using a CCM-220 Chlorophyll Content Metre (Opti-science which uses absorbance to estimate the chlorophyll content in leaf tissue. The metre measures the absorbance of both wavelengths and calculates a CCI (chlorophyll content index) value that is proportional to the amount of chlorophyll in the sample, with higher values indicating more chlorophyll. A mean of three measurements per tree (a high, a middle and a low leaf) were analysed.
Results
At the time of release the temperature was over 12°C and by the 24 hr assessment all aphids had flown from the container. The mean number of aphids on the Bramley, Ontario and Priscilla cultivars were 5.6, 2.6 and 3.2 respectively (Fprop 0.324, lsd 4.538, sed 1.968, 8 d.f.) although this was not statistically significant. Leaf colour was different between cultivars with Ontario having a higher CCI reading than Bramley and Priscilla, 40.1, 29.6 and 30.3 respectively (Fprop 0.052, lsd 9.11, sed 3.95, 8 d.f.), although there was variation between the leaves at different levels.
It appears gynoparae land on all cultivars and remain once on the plant (one Ontario that had eight aphids landing on 9 November, still had six by 12 November). This would imply the resistances are not antixenotic (deter the aphids from staying), but rather antibiotic (affect the aphids performance).
4.3.4 Effect of apple variety on aphid weight
Methods
The effect of apple variety on aphid weight was assessed for seven cultivars: Ontario, Saturn, Goldrush, Greensleeves, Liberty, Priscilla and Florina. Young potted apple seedlings were placed in a gauzehouse in a randomised block design, with at least five replicates of each variety, and six of Priscilla and Greensleeves.
Bramley apple seedlings were inoculated with adult RAA and were allowed to deposit nymphs. The adults were then removed. Five-day old nymphs were weighed on a Cahn electrobalance in the laboratory, and were placed in individual micro-centrifuge tubes and then transferred to the gauze-house. Each apple seedling was inoculated with one aphid on an upper leaf. The leaf was covered with a bread bag secured with a wire at the base. Aphids were allowed to settle and to feed for 3 days before being reweighed. Again they were transported in micro-centrifuge tubes held in a cool box, and aphids were removed from the tube for weighing. The experiment was repeated with a second set of aphids once the first set had been removed. These were introduced to a different leaf on the tree.
Results
Apple cultivar had a significant effect on weight loss in RAA (Table 4). This was most obvious for Ontario, where every aphid had a reduction in weight.
Table 4. The effect of apple variety on weight (milligrams) production of RAA nymphs


Cultivar

Mean weight change

(+ or –)

mg per aphid

S.E.

Number of aphids weighed and included

Number of aphids missing or dead

Ontario

-0.0307

0.01507

6

4

Saturn

-0.0064

0.01167

10

1

Goldrush

-0.0041

0.01309

8

2

Greensleeves

0.0134

0.01171

10

2

Liberty

0.0215

0.01171

10

0

Priscilla

0.0370

0.01305

8

4

Florina

0.0729

0.01171

10

0



4.4 Monitoring aphid distribution on flowered plants
After the first year’s gauzehouse host alternation sampling was completed, spare replicates of cultivars in good condition were put into the gauzehouse for the aphids to return to from plantain, mate and lay eggs. This was done to determine if any of the cultivars resisted the returning aphids in autumn. It has been reported that several defence related genes, such as PR proteins are known to be upregulated during leaf senescence, so these could influence reinfestation levels.
These trees were left unpruned and so produced floral buds with associated spur leaves, more like commercial tree architecture. Trees were watched closely to see where the aphids fed in relation to the developing flowers.
Table 5 . Aphid feeding locations on flowering apple trees


Cultivar

Location of aphids

Comment

1

Bramley

L + F




2

Discovery

L + F




3

Florina

L + F




4

GoldRush

F only

Avoided leaves

5

Greensleeves

L + F




6

Liberty

F only

Avoided leaves

7

Malus floribunda

F only

Avoided leaves

8

Malus robusta

No spare plants




9

McIntosh

L only

Flowers untouched

10

Ontario

L + F




11

Priscilla

No flowers (on leaf stalks only)

Avoided leaves

12

Saturn

L + F




L = Leaf; F = Flower
All the plants developed RAA in the spring, showing, at least in the confines of a small gauzehouse, RAA lands on all the cultivars to breed.
In all the plants, some aphids were seen feeding on the flower stalks, with the exception of McIntosh where the aphids remained on the leaves. The cultivars scored as resistant before (section 4.1.3) because the aphids stayed on the stems (Goldrush, Liberty and M. floribunda) away from the leaves, now show this trait to be a negative one as it forces the aphids onto the flower stems. McIntosh on the other hand, because of it susceptible leaves, does not. Why McIntosh is the only cultivar not to have aphids on the flower stalks is not clear, and needs to be confirmed with more samples. If borne out it is possible this might mean that the aphid does not cause fruit damage, despite infesting the leaves. Briggs and Alston (1969) noted that three siblings of McIntosh were infested but showed no apple damage. This possible tolerance effect to RAA warrants further investigation.
4.5 Aphids observed on field grown apples
In 2006, RAA numbers were particularly high in apple orchards. This provided a good opportunity to rescore a plot of 28 cultivars for RAA incidence used in previous varieties trial.
Table 6 is an edited version of a table from that trial (Cross et al., 2005 – table 39) and shows the 2006 scores to be substantially higher than the earlier observations in 2004.
This table highlights how the low incidence and patchy distribution of RAA in orchards can lead to erroneous appraisals of resistance. For example, the infestation levels of 2004 lead to the erroneous conclusion that the cultivars Red Falstaff (grafted) and Rubinola are resistant. The 2006 scores now show them to be highly susceptible. The table does, however, support the inoculation results from this project’s inoculation and the conclusion that cvs Liberty, Goldrush and Delorina are very strongly resistant and that Florina is highly susceptible. Goldrush was badly infested with mildew, despite being listed as being resistant to mildew (see Annex 1).

Table 6. Incidence of RAA scores on a multi-cultivar apple planting at EMR carried out in 2004 and 2006


Cultivar

2004

2006

Ariwa

Bohemia


Ceeval

D3

Delorina



Discovery

DL11


Ecolette

Ed VII


Encore

Florina


Goldrush

Howgate


Judeline

Liberty


Pikant

Pilot


Pinova

Rajka


Rebella

Red Falstaff

Red Falstaff g.

Resi


Rubinola

Rubinstep

Santana

Topaz


Worcester Permain

2

2

5



2

0

4



3

3

2



1

6

0



5

2

0



2

9

7



2

7

6



0

1

0



9

1

4



3

13

16

13



12

0

13



9

13

5



7

15

0



12

8

0



9

15

13



9

13

13



8

6

11



14

7

13



12



5 GENE EXPRESSION PROFILING
5.1 Differential display reverse transcription (DDRT)-PCR
The experimental design employed had many safeguards against false positives, namely multiple RAA clones and multiple cultivar clones, run with multiple differential display kits. Each gel was run with as many replicates as is possible and with never less than three.
Twenty-four arbitrary 13mers were used (three RNAimage Kits; 1, 5 and 6) during the life of this project giving an estimated 60% probability of detecting any given mRNA.
Total RNA was isolated in batches of four aphids from the same plant, to maximise quality/quantity of RNA and obtain a more representative snapshot of aphid expression. As RNA is very prone to degradation the samples were kept frozen or in protective buffers at all times. The aphids were homogenised whilst still frozen by vigorously agitating them in a tube with a ball bearing for 2 minutes on a mixer mill cooled with liquid nitrogen. The homogenate was extracted with an RNeasy extraction kit (Qiagen). The RNA was then treated with RNase-Free DNase Set (Qiagen) to remove all traces of DNA, eluted with 50 ul water and then immediately frozen and stored at -80°C.
mRNA from each sample was reverse transcribed into cDNA as three populations using three one-base-anchored oligo-dT primers and reagents in the RNAimage Kit (BioGene). Each of these three cDNA types were then used as templates for eight PCR reactions, using the same oligo-dT primer used previously and one of eight arbitrary 13mers provided in a RNAimage Kit. Radioactive alpha-[33P]dATP was included in the PCR so that all products were radiolabelled. PCR products (each representing an expressed gene) were then separated on 5% denaturing polyacrylamide gels which were then dried and bands visualised on X-ray film.
When the film was placed on top of the dried gels, it was secured firmly down with tape to stop any movement. A pin was then used to punch multiple holes through the film and into the gel. When differentially expressed genes were discovered and needed to be excised from the gels for sequencing, the film could be perfectly realigned with gels by aligning all the holes.
Bands were excised using a clean razor blade and placed in 0.5 ml 2X PCR buffer to elute the DNA with minimum degradation. Each band was reamplified using the original PCR primers, cloned using a pGEM-T vector kit (Promega) and sequenced.
5.2 Aphids sampled from apple cultivars of high and low susceptibility
For the susceptible/resistant cultivar profiling, each differential display reaction was initially run in blocks of nine co-run samples. Here the samples were a RAA clone from three susceptible cultivars (Saturn, Discovery, Bramley), five resistant cultivars (Ontario, Priscilla, M. robusta, Goldrush, Liberty) and a starved aphid. A starved control was used to produce a comparative gene expression profile of an aphid that is under-nourished rather than being under chemical stress from the apple cultivar.
It was apparent that each of the ‘resistant’ cultivars was likely to be using a different resistance mechanism to combat RAA. Therefore, differentially expressed bands may not be consistent between cultivars, and so do not facilitate the criteria for avoiding cutting out false positive bands (i.e. the bands must be consistent across replicates). So rather than running all ‘resistant’ cultivars together the focus was turned to the only consistent resistant cultivar, Ontario. Gels were now run in blocks of six (three samples from different susceptible cultivars and three samples from different Ontario replicates). Now only bands that were present in all the susceptible cultivars, or in all the Ontarios, were characterised. This dramatically improved the confidence in interpreting gels and number of bands that could be excised.
5.3 Aphids sampled during host alternation
For the gene expression analyses leading up to and after host alternation, each reaction consisted of 12 co-run samples: Five replicates of RAA clones early in the season (May/early June) compared with late in the season (late July), and two from plantain (October).
The cultivars used were Greensleeves (potted), Florina (potted and orchard), Red Falstaff (orchard), Discovery (potted and orchard), Ecolette (orchard), Red Pippin (orchard).
Only gel bands were sought that were unique to all the early season or late season aphids, and unique to the apple or plantain living aphids.
6. CHARACTERISATION OF DIFFERENTIALLY EXPRESSED GENES
6.1 Sequence acquisition
Once sequenced, each sequence was trimmed to remove plasmid and checked for the presence of the expected random and anchored primers. Without exception, every sequence had the expected random primer at one end and the anchor primer at the other.


    1. Database matching

All the sequences were tested for database matches using the NCBI online BLAST package (http://www.ncbi.nlm.nih.gov/blast/Blast.cgi).


Firstly, each sequence was tested for a match to any aphid sequences in the nucleotide sequence databases using the BlastN. The best matches are shown in Annex 2, column 5. As these sequences are biased to the 3’ end of mRNAs, the matches to the EST database often provided valuable extra sequence for eventual gene identification, particularly for the shorter sequences. This negates the need to resort to complicated RACE (rapid amplification of cDNA ends) protocols.
The identity of the genes were explored using BlastX against the peptide sequence databases SwissProt and nr (all non-redundant GenBank CDS translations + RefSeq Proteins + PDB + SwissProt + PIR + PRF). Amino acid matching (instead of DNA) needed to be performed because amino acid sequence evolves much slower than DNA, thus increasing the probability of finding matches with one of the organisms in the databases.
In all, 88 clones were sequenced and are catalogued in Annex 2, along with the function of those that could be identified.
6.3 Genes discovered
A comprehensive list of all the differential bands cloned, sequenced and characterised is given in Annex 2. Tables 7 and 8 show a simplified list of the genes with their biological functions.
6.3.1 Genes differentially expressed on susceptible and resistant apple cultivars and subsequent insights
The genes characterised are involved in a number of biological systems, although mainly protein synthesis and energy metabolism. Thus, a major global metabolic change appears to occur on aphids living on the resistant apples.
Disruption of the energy metabolism pathways would naturally have a dramatic effect on the aphid’s survival. NADH:ubiquinone dehydrogenase is seen to be down regulated in the resistant samples. This enzyme is the first enzyme of the mitochondrial electron transport chain. It is the well known as being the target for the organic insecticide Rotenone found in several plants. This suggestion correlates well with the effects seen on aphids on the resistant plants.
A decline in protein synthesis would also result from the three down regulation synthesis genes, which eventually deprive the aphids of fundamental enzyme activity.
Table 7. Genes differentially expressed by RAA living on susceptible and resistant apple cultivars


Susceptible

Resistant

Gene

Function

Gene

Function

ATP-dependent RNA helicase

Protein synthesis

Cuticle protein

Structure

Ubiquitin ligase E3

Metabolism

Legumain-like protease

Digestion

Cold shock domain-containing protein E1

Protein synthesis

Picorna-like virus

Polyprotein



Virus

Elongin c

Protein synthesis

Troponin I

Muscle contraction

CUB domain-containing protein

Development?

Heterogeneous nuclear ribonucleoprotein

RNA transport

Buchnera heat shock protein dnaK

Stress/chaperone

Histone H3 family 3B

Structure/expression

NADH:ubiquinone dehydrogenase

Energy metabolism

Apolipophorin

Lipid transport

Calmodulin

metabolism

UNC-13-B protein

Synaptic transmission

Heat shock protein 70

Stress/chaperone

Transcription initiation factor TFIID subunit

Protein synthesis

Steroid dehydrogenase

Hormone metabolism

Baculovirus Late expression factor 4

Virus







Citrate synthase

Energy metabolism







Chromatin associated protein KTI12

Protein synthesis







Fatty acid desaturase

Fatty acid metabolism







Mitochondrial ATP synthase (c subunit)

Energy metabolism







60S ribosomal protein L23 (L17A)

Structure/expression







small heat shock protein hsp21.4

Stress/chaperone







Vacuolar protein sorting-associated protein 26

Protein trafficing

Elongation factor 1-alpha

Protein synthesis

Elongation factor 1-alpha

Protein synthesis

A surprising result is that the heat shock protein 70 and its prokaryotic homologue in the symbiont Buchnera (heat shock protein dnaK) are both detected in the susceptible aphids. Intuitively it might be expected to see these two major stress proteins upregulated in the stressed resistant aphids. This suggests these genes are inhibited in the resistant samples which may lead to protein instability in the aphids.


Elongation factor 1-alpha which is involved in the binding of aminoacyl-tRNAs to ribosomes during protein synthesis occurs multiple times in all samples except the plantain ones. This gene primes off all three anchor primers and many random primers. It is difficult to determine if it has any significance being so ubiquitous. There does however seem to be at least two paralogues that have been discovered in holometabolous insects.
Cuticular proteins were also well represented in the resistant samples, varying in size on gels due to multiple repeats within the gene. Their appearance and upregulation in resistant samples is unclear.
There has also been an unexpected discovery of two viruses infecting RAA (Ryabov, 2007); these may be activated by the aphid’s stress. The effects of these viruses on RAA require further investigation.
Some of the genes discovered here were differentially represented in a recent proteomic study of Myzus persicae (Francis et al., 2006) during a host switch. In their study, 14 aphid proteins were found to vary according to host plant switch, ten of them were down regulated (proteins involved in glycolysis, TCA cycle, protein and lipid synthesis) while four others were overexpressed (mainly related to the cytoskeleton).
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