JXB Advance Access originally published online on May 23, 2006
Journal of Experimental Botany 2006 57(10):2249-2257; doi:10.1093/jxb/erj192
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© 2006 The Author(s).
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.This paper is available online free of all access charges (see http://jxb.oxfordjournals.org/open_access.html for further details)
RESEARCH PAPER |
Effect of eyespot caused by Oculimacula yallundae and O. acuformis, assessed visually and by competitive PCR, on stem strength associated with lodging resistance and yield of winter wheat
Crop and Environment Research Centre, Harper Adams University College, Newport, Shropshire TF10 8NB, UK
*To whom correspondence should be addressed. E-mail: rray{at}harper-adams.ac.uk
Received 2 September 2005; Accepted 13 March 2006
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Abstract |
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Winter wheat, (cv. Consort) was inoculated with three isolates of either Oculimacula yallundae or O. acuformis to determine the effect of eyespot caused by each species on yield and lodging resistance of winter wheat. Plants were visually assessed for disease incidence and severity, and pathogen DNA was quantified at GS 33 and GS 60. At early milk development of the crop (GS 72), 900 main shoots were also visually assessed for the disease and subjected to mechanical tests for stem strength. Pathogen DNA was extracted from each shoot and quantified using competitive polymerase chain reaction (PCR). Although slight and moderate eyespot lesions caused by either species had no effect on ear weight, severe lesions caused by O. acuformis and O. yallundae reduced ear weight by 3% and 7%, respectively. Stem lodging failed to occur at the site; however, yield losses of 11% for O. acuformis and 6% for O. yallundae were observed. Visual assessment failed to reveal differences between species in their effect on plant characteristics, stem bending strength, or stem safety factor. PCR data, however, showed that the two species had similar effects determined by different DNA concentrations. Both species reduced lodging resistance (stem safety factor) compared with the control. In contrast to healthy plants, where reductions were related predominantly to the height and weight distribution of the plants, the observed reductions of stem lodging resistance in infected plants with Oculimacula spp. were associated primarily with reductions in stem bending strength.
Key words: Eyespot, lodging, Oculimacula spp., PCR, winter wheat, yield
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Eyespot caused by Oculimacula yallundae and O. acuformis (Robertse et al., 1995; Crous et al., 2003) is considered the most important stem base disease of cereals in temperate countries. Yield losses due to the disease on winter wheat are associated with direct effects of moderate or severe lesions, interfering with the movement of nutrients and water through the stem, and the effects of severe lesions causing lodging (Glynne and Salt, 1958; Scott and Hollins, 1974; Clarkson, 1981). Winter wheat grain losses of up to 33% in the absence of lodging have been reported following artificial inoculation (Oort, 1936; Glynne and Salt, 1958). Moderate or severe eyespot lesions have been shown to reduce thousand grain weight by up to 5% and 15%, respectively (Defosse and Rixhon, 1968; Scott and Hollins, 1974; Clarkson, 1981). The ultimate effect of severe eyespot lesions, however, is the weakening of the stems to such an extent that they collapse in all directions (Butler, 1957). In comparison, stem lodging (stems breaking at their internodes) or root lodging (stems rotating about their base) caused by natural forces occur in a uniform direction across the field (Pinthus, 1973). Many of the cultural field practices as well as environmental conditions conducive to natural lodging are also conducive to eyespot disease. For example, early sowing (Colbach and Saur, 1998) and abundant moisture supply have been shown to favour both eyespot and natural lodging (Pinthus, 1973). It is unclear, therefore, if the correlation between eyespot disease and lodging is because of positive effects of eyespot on lodging or the effects of certain plant characteristics or environmental factors influencing both.
Crook and Ennos (1994) investigated the mechanical basis of stem lodging in winter wheat cultivars and determined that their resistance to simulated lodging was related more to the stem characteristics of the plants than to the bending strength or rigidity of the stem. Natural stem lodging occurred during grain filling when wheat ears were heaviest and the stem failed to support the overturning moment generated by the ‘self-weight’ of the plant (stem height and weight, including the ear). Thus, lodging resistance was associated with shorter and lighter plant shoots. The outcome of severe eyespot on winter wheat, however, is the reduction of ear weight (Scott and Hollins, 1974). Severely infected plants would, therefore, be expected to have much lighter shoots than uninfected plants. In these terms, it could be assumed that the lodging resistance of eyespot-infected plants would be higher as a result of reduced stem weight. Eyespot-induced lodging has, however, been shown to occur in such lighter crops and has been attributed to the severe infection rotting the stems. Indeed, Glynne and Salt (1958) reported that an increase from 35% to 85% of stems with severe eyespot had an effect on lodging similar to an increase in stem weight from 5 to 7.5 t ha–1.
The aims of this study were to establish the effect of eyespot caused by O. yallundae or O. acuformis on the yield of winter wheat and to identify the mechanism of eyespot-induced lodging using mechanical tests. Differences between species causing eyespot and disease effects on lodging resistance or yield were determined using visual assessment and competitive polymerase chain reaction (PCR) assays.
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Materials and methods |
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Field operations
To minimize the risk of naturally occurring eyespot, a field site at Tibberton, Shropshire was selected because it had had a 9 year break from cereal production. The winter wheat cultivar Consort was sown on 2 November 2001 at a rate of 356 seeds m–2 following potatoes. The experiment was set up as a randomized block design with eight 1.8x12 m replicated plots for each treatment. Plots were artificially inoculated with three individual isolates of O. yallundae or O. acuformis, or left untreated (control). A guard strip of 0.5 m between each plot was left in order to reduce the movement of inoculum between plots. To promote conditions conducive to lodging, plant growth regulators were not applied to the crop. Early in the season, poor plant growth up to stem extension (GS 31; Zadoks et al., 1974) was observed due to manganese deficiency and weed infestation on the site. Consequently, five applications of micronutrients and nitrogen fertilizer were applied during spring. Ally (20 g kg–1 metsulfuron-methyl) and Starane (200 g l–1 fluroxypyr) were applied to the crop at full rates. Pressure from other cereal diseases was low throughout the season. However, one application of Folicur (250 g l–1 of tebuconazole), Tern (750 g l–1 of fenpropidin) and Amistar (250 g l–1 azoxystrobin) at field rates of 0.4, 1, and 0.5 l ha–1, respectively, was made following stem extension.
Plots were harvested using a small plot combine harvester (Winterstieger Crop Master), and grain yield (at 15% moisture content), thousand grain weight, and specific (hectolitre) weight were determined.
Isolates of Oculimacula spp. used, inoculum preparation, and application
Single spore isolates of O. yallundae and O. acuformis used for the artificial inoculation of wheat plants were taken from sporulating colonies from each pathogen obtained from the culture collection at Harper Adams University College (Table 1). The selected isolates were grown on potato dextrose agar at 20 °C for 5 weeks. The method for production of oat-grain inoculum was modified from Bruehl and Nelson (1964). Distilled water (240 ml) was added to oat grains (300 g) and the mixture was autoclaved for 1 h on two consecutive days. Five mycelial plugs (5 mm diameter) from each isolate were placed in each bag of sterile oats. Incubation took place at 20 °C for 
6 weeks. The inoculum was applied on the field manually at a rate of 6 g m–2 when the seedlings had two leaves unfolded on the main shoot (GS 12; Zadoks et al., 1974). To determine pathogen biomass in the inoculum, following inoculation, pathogen DNA present in oat-grain inoculum was extracted from one sample of 10 g of oats from each thoroughly mixed oat-grain inoculum of each isolate. Pathogen DNA (Table 1) was quantified using competitive PCR assays as detailed previously (Ray et al., 2004).
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Sampling and disease assessment
Disease assessments for eyespot were carried out on 30 plants per plot collected at stem elongation, when the third node was detectable (GS 33), and on 30 main shoots at anthesis (GS 60) from each plot as detailed previously by Ray et al. (2004). Immediately following assessment, pathogen DNA was extracted from plant material and quantified using competitive PCR (Ray et al., 2004). At GS 33, single shoots and tillers of five plants per plot were counted, weighed, and assessed for eyespot on a scale of 0–3, where 0 was not infected and 3 was severely infected. Then, a disease index (DI) was calculated (Scott and Hollins, 1974) for each plant and averaged for each plot.
Single plant shoots at GS 72 and assessment of disease and lodging resistance
At early milk development of the crop (GS 72), 15 plants were collected at random from each plot. The main shoot from each plant was separated manually from the tillers and kept in a plastic bag in a cold room at 4 °C to minimize loss of fresh weight and pathogen DNA accumulation. Assessments of 900 single main shoots were made within 72 h of sampling. Height, fresh weight, and centre of gravity were recorded for each shoot as described previously by Crook and Ennos (1994). The centre of gravity was determined by placing each shoot across an outstretched index finger and moving the shoot along the finger until the balance point was reached. The height of the centre of gravity was the distance from the base of the stem to the balance point. The ear of each shoot was removed and weighed and the shoot was then assessed for disease severity (Scott and Hollins, 1974).
Mechanical tests on stems
Stem lodging occurs naturally when the stem cannot support the overturning moments generated by the self-weight of the plant (Crook and Ennos, 2000). The lodging meter used in this study (Crook and Ennos, 2000) measures the bending strength of the stem. The basal 7 cm length of each shoot was cut and placed (eyespot lesions always facing the same direction) in the holding cup (1 cm deep) of the lodging meter and the lodging arm was rotated until the stem buckled (Crook and Ennos, 2000). Failure occurred at the eyespot lesion, which was usually at 2–4 cm from the base of the stem. The maximum force required to buckle the stem, thus bending strength (Nm), was recorded for each shoot. The self-weight moment (M) of each main shoot at 30° from the vertical was calculated using Equation 1.
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DNA extraction and quantification of single shoots
Following mechanical tests, the same stem cuttings were chopped finely and freeze-dried for DNA extraction. Each shoot was placed in a 57 mm x 15.3 mm length plastic tube (Sarstedt Ltd, Leicester, UK) together with three steel ball bearings (8 mm diameter), and homogenized using a soil mill for 1–2 h. DNA extraction was carried out as described previously (Ray et al., 2004), except that all volumes were adjusted for the smaller volume of plant material. Total DNA concentration was determined and competitive PCR performed on DNA extracted from each shoot as described by Ray et al. (2004).
Statistical analysis
All data were analysed using analysis of variance and regression analysis using Genstat® Version 4.1 for Windows (Lawes Agricultural Trust, UK). Where necessary, DNA and DI or incidence data were transformed using log10 and angular transformations, respectively, in order to normalize the frequency distributions. Data from single shoot assessments were separated and averaged according to an eyespot score of slight, moderate, and severe for each treatment. Multiple linear regressions with groups were used to analyse relationships between variables for the two Oculimacula spp.
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Results |
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Disease incidence, severity, and DNA accumulation for O. acuformis and O. yallundae
No significant differences were observed between isolates within species, and all caused eyespot disease with similar incidence and DI at each growth stage. At GS 33, plants inoculated with isolates of O. yallundae had significantly higher eyespot incidence and DI (assessed using 30 plants, or five plants and tillers) than plants inoculated with isolates of O. acuformis or the control (Table 2). The incidence of eyespot at GS 33 was 35.2% and 68.9% for plots inoculated with O. acuformis and O. yallundae, respectively. At GS 60 and 72, although no significant differences were observed between plants inoculated with O. acuformis or O. yallundae, the control plants had significantly lower eyespot incidence and DI than plants inoculated with either species.
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DNA concentrations measured at each growth stage are presented in Table 3. PCR quantification revealed that DNA of both species was present as early as GS 33 in the control (1.10 and 0.23 pg ng–1of total DNA for O. yallundae and O. acuformis, respectively). Mean background DNA found in inoculated plots was 0.78, 2.9, and 2.6 pg ng –1 of total DNA at GS 33, 60, and 72, respectively. No significant difference was found between isolates within each species for DNA concentrations measured from bulk extractions at GS 33 and 60. Pathogen DNA in the control plants increased at GS 60 and GS 72 to 4.1 and 31.7 pg ng–1 for O. yallundae, and 8.1 and 9.0 pg ng–1 for O. acuformis, respectively.
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At GS 33, plants inoculated with O. acuformis had nearly 50% less pathogen DNA present in their stems than those inoculated with O. yallundae (Table 3). By GS 60, pathogen DNA in the plants inoculated with O. acuformis was significantly higher than in those inoculated with O. yallundae, in which concentrations of O. yallundae DNA were similar to DNA concentrations of both species found in the control plots. The reverse result was obtained for DNA quantified from single stem extractions at GS 72, where O. yallundae was found in five times higher concentrations than O. acuformis (Table 3). Total (plant and fungal) DNA quantification of single shoot DNA at GS 72 revealed that shoots inoculated with O. yallundae had 25–50% lower total DNA concentrations than shoots inoculated with O. acuformis. Indeed, it was noted during eyespot assessments at GS 72 that many of the main shoots infected with O. yallundae had much more straw-like appearance compared with shoots infected with O. acuformis or the control.
Plant stem characteristics, lodging resistance, and yield
Neither root nor stem lodging occurred at the field site. No significant differences were observed between plots inoculated with the two Oculimacula species for any of the plant characteristics or yield measured in this study. At GS 33, eyespot caused by either species had no significant effect on fresh weight of tillers (4.01, 4.05, and 3.86 g for the control, O. yallundae-inoculated, and O. acuformis-inoculated plants, respectively). However, at the same growth stage, a significant reduction in tiller number was observed for plants inoculated with either species compared with the control (Table 4). At GS 72, disease effects on tiller height, ear weight, or centre of gravity averaged per plot were not significant (Table 4). However, it must be noted that by GS 72, high concentrations of DNA of both species were found in the control plots; thus the comparison was limited due to the lack of disease-free plants. Even under such circumstances, significant differences between inoculated and non-inoculated plots were found for stem bending strength, safety factor, and yield. Stems with moderate lesions contained 27 and 157 pg ng–1 DNA of O. acuformis and O. yallundae, and reduced stem safety factor by 13% and 14% compared with the control, respectively (Table 5). Severe lesions corresponded to 40 and 234 pg ng–1 DNA of O. acuformis and O. yallundae and caused reductions of stem safety factor of 36% and 33%, respectively. Although slight and moderate lesions of both species failed to show an effect on stem strength, severe lesions reduced stem bending strength by 35%. At harvest, plants inoculated with O. acuformis had significantly lower grain yield than the control (Table 4). Oculimacula acuformis reduced overall yield by 11%. Although smaller yields were also measured from plots inoculated with O. yallundae, overall yield was not significantly different from the control.
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Significant differences were observed between isolates of O. acuformis for DNA concentrations at GS 72, tiller weight, and self-weight moment (Table 6), and between isolates of O. yallundae for background DNA of O. acuformis, bending strength, and safety factor (Table 7). Isolates of O. acuformis with lower DNA concentration at GS 72 caused less reduction in tiller weight and self-weight moment. Isolates of O. yallundae showed a greater effect on stem bending strength and safety factor where lower concentrations of background DNA of O. acuformis were found.
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Relationships between pathogen DNA, grain yield, and plant characteristics associated with stem lodging resistance
Although a significant negative relationship was observed between DNA of O. acuformis at GS 33 and yield, it accounted for only 9% of the variance [yield (t ha–1)=–0.41 (log10 DNA of O. acuformis at GS 33)+7.50, P=0.014]. The same regression analysis performed using DNA of O. acuformis at GS 72 found in all main shoots, categorized as slight, moderate, and severe, revealed a similar relationship [yield (t ha–1)=–0.42 (log10 DNA of O. acuformis at GS 72)+7.88, P <0.001], accounting for 38% of the variance. There were no significant relationships between yield and DNA of O. yallundae at any growth stage.
Regressions of self-weight moment, stem safety factor, and stem bending strength on eyespot scores at GS 72 fitted a common line for both Oculimacula spp. (Fig. 1A–3A23A). However, regressions of those characteristics on DNA concentration of the two fungi fitted two separate lines. The lines were parallel, with negative slopes for self-weight moment (Fig. 1B) and safety factor (Fig. 2B), indicating that relatively more DNA of O. yallundae was required to achieve the same effect as O. acuformis. Regression of bending strength on pathogen DNA at GS 72 showed that the species fitted two non-parallel lines, accounting for 61% of the variance (Fig. 3B).
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The reduction in stem safety factor for all inoculated plots was associated more with reduction in stem bending strength, accounting for >85% of variance, than with reduction in self-weight moment of the plants.
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Discussion |
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The main findings of this work are that O. acuformis can cause severe yield loss of winter wheat and that effects on plant characteristics associated with lodging of both species are similar but dependent on different pathogen DNA concentrations. Both species demonstrated an ability to reduce the stem safety factor by reducing stem bending strength. In comparison, the safety factor for uninfected crops to naturally occurring lodging would be largely dependent on the self-weight moment of plants (Crook and Ennos, 1994). Severe eyespot caused by both species can increase the risk of stem lodging by reducing stem strength significantly. The effects on stem lodging and plant characteristics associated with it may also be influenced by the aggressiveness of individual isolates within the species. Thus, some of the isolates of O. acuformis and O. yallundae may be more damaging than others. The significant isolate variation found within O. acuformis and O. yallundae at GS 72 suggests that for inoculated experiments, the selection of isolates for eyespot inoculum is important in order to obtain representative results.
Slight and moderate eyespot lesions caused by the species had no effect on ear weight as indicated by the analysis of averaged values per plot. However, severe lesions caused by O. acuformis and O. yallundae reduced ear weight by 3% and 7%, respectively, corresponding to >60% more pathogen DNA concentration than in the control. This is in agreement with previous reports that losses measured on single stems are mainly due to severe lesions and not to slight or moderate lesions caused by Oculimacula spp. (Jørgensen, 1964; Scott and Hollins, 1974).
The observed reduction in tiller number in inoculated plots at GS 33 supports early reports on this effect of severe eyespot on winter wheat (Sprague and Fellows, 1934; Bruehl et al., 1968; Scott and Hollins, 1974).
Previous studies (Glynne et al., 1945; Scott and Hollins, 1974) have demonstrated that results derived from single stem observations may not represent the actual effect of the disease on the whole crop or plants where compensation may occur from the less diseased plants and tillers. In this study, such compensation effects were not observed for O. acuformis, which reduced yield by 11%. Although plots inoculated with O. yallundae had 6% less yield than the control, the difference was not statistically significant. The large differences between species in their DNA concentrations at GS 60 indicated that the disease developed more rapidly in the O. acuformis-inoculated plots. Therefore, it is possible that infection by O. acuformis had spread onto more tillers than O. yallundae, where yield loss may have been partially compensated by the less-infected neighbouring tillers. The yield losses of 11% for O. acuformis and even 6% for O. yallundae are in agreement with losses of 11–12% and 7–10% for eyespot in inoculated experiments in the absence of lodging reported by Scott and Hollins (1974) and Jørgensen (1964), respectively.
Visual assessment using 30 plants or using just five plants with all tillers provided similar results, indicating that under high disease pressure, sample size is less critical due to a more even spread of the disease. The observed differences in eyespot incidence and severity at GS 33 were consistent with previous reports that the incidence and severity of eyespot symptoms at the early crop growth stages are greater when the disease is caused by O. yallundae (Bateman, 1990; Goulds and Fitt, 1991a, b). The higher incidence and DI of plants infected with O. yallundae also corresponded to the larger concentrations of pathogen DNA.
The absence of marked differences between species for disease incidence or severity late in the season has been found in other studies (Goulds and Fitt, 1988). Similarly, in this study, visual assessement failed to determine differences between species at GS 60 and GS 72. However, DNA quantification showed that different concentrations of the two species caused visually similar symptoms. The DNA of O. acuformis increased 10-fold from GS 33 to GS 60 and remained at this concentration at GS 72. In comparison, DNA of O. yallundae increased only 2-fold up to GS 60, but then increased >10-fold by GS 72. One reason for this increase at GS 72 could be because pathogen DNA is measured against total DNA, and thus decreases in plant DNA are reflected as relative increases in pathogen DNA. Indeed, the plants infected with O. yallundae had a more profound straw-like colour associated with lignified, mature tissue, and quantification of total DNA showed it to be up to 50% less than total DNA extracted from O. acuformis-infected plants. Increased lignification and cell wall thickening at GS 71 of several winter wheat cultivars, as a reaction to eyespot stem infection, have been reported (Murray and Bruehl, 1983), although it is unclear from their work which Oculimacula sp. was used for inoculation in the experiment.
Analyses of position and parallelism using visually assessed eyespot score data at GS 72 failed to reveal any differences between species in their effect on plant characteristics, stem bending strength, or safety factor. Use of DNA quantification data, however, showed that the two Oculimacula spp. fitted, on most occasions, parallel lines, and had similar effects determined by different DNA concentrations. Crook and Ennos (1994) showed that resistance of modern winter wheat cultivars to naturally occurring stem lodging was not primarily related to the strength of the stems, but rather to their height and weight distribution. In this study, both species reduced the stem lodging resistance (stem safety factor) compared with the uninoculated control. The observed reduction in stem safety factor was associated mainly with the reduction of stem bending strength observed for the shoots from inoculated plots (R2=0.85, P <0.001). The main difference between the species was related to their effect on stem bending strength, and analyses of position and parallelism suggested that the data were best fitted by two separate lines with different slopes and intercepts for each species. Although regression analysis indicated that less pathogen DNA of O. acuformis than of O. yallundae was necessary to reduce stem bending strength, the steeper line of the latter suggested that reductions caused by this pathogen will occur over smaller DNA increases.
Regressions of stem bending strength on DNA of O. yallundae accounted for >70% of the variance compared with O. acuformis, which accounted for 50%, indicating that other factors also contributed to this effect. One possible factor was the variation in DNA concentration among isolates of O. acuformis at GS 72. This is highly relevant since the differential effect of the species on stem bending strength was related to the DNA concentration of their isolates at GS 72. Poupard et al. (1994) also reported significant variation between individual isolates of the two Oculimacula spp. in their development in wheat plants at different growth stages as measured by enzyme-linked immunosorbent assay (ELISA). Differences between individual isolates of O. acuformis were also observed for tiller weight and self-weight moment. Isolates with greater DNA concentrations appeared to reduce tiller weight and self-weight moment of the stems more. Individual differences between isolates of O. yallundae in stem bending strength and safety factor against stem lodging were associated with different concentrations of background DNA of O. acuformis detected on the same shoots. Reductions in stem bending strength and safety factor were greatest on shoots inoculated with individual O. yallundae isolates where less background DNA of O. acuformis was detected. Since no difference between isolates of O. yallundae for DNA concentration at GS 72 was found, the exact reason for the observed differences is unclear, but it is possible that some isolates were less aggressive and more tolerant of secondary infections by isolates of O. acuformis. Thus, the overall effects of each Oculimacula sp. on those plant characteristics and factors associated with lodging, where significant isolate variation was found, were determined by the intinsic aggressiveness of individual isolates within the species. To clarify these effects, a larger number of isolates would be required in further work.
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Acknowledgements |
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We would like to thank Syngenta Ltd for funding, Dr Jim Beck from Syngenta Biotech. Inc., USA for supplying the primers, Dr Geoff Bateman from Rothamsted Research for his helpful advice, and the trial officers at the Crop and Environment Research Centre at Harper Adams University College for technical assistance with the trial.
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Footnotes |
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Present address: Air Pollution Group, Westlakes Scientific Consulting Ltd, Westlakes Science and Technology Park, Moor Row, Cumbria CA24 3LN, UK 
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References |
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