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1 New Zealand Journal of Agricultural Research ISSN: (Print) (Online) Journal homepage: Impact of clover root weevil Sitona lepidus (Coleoptera: Curculionidae) larvae on herbage yield and species composition in a ryegrass white clover sward P. J. Gerard, D. L. Hackell & N. L. Bell To cite this article: P. J. Gerard, D. L. Hackell & N. L. Bell (2007) Impact of clover root weevil Sitona lepidus (Coleoptera: Curculionidae) larvae on herbage yield and species composition in a ryegrass white clover sward, New Zealand Journal of Agricultural Research, 50:3, , DOI: / To link to this article: Published online: 23 Feb Submit your article to this journal Article views: 192 View related articles Citing articles: 10 View citing articles Full Terms & Conditions of access and use can be found at

2 New Zealand Journal of Agricultural Research, 2007, Vol. 50: /07/ The Royal Society of New Zealand Impact of clover root weevil Sitona lepidus (Coleoptera: Curculionidae) larvae on herbage yield and species composition in a ryegrass-white clover sward P. J. GERARD D. L. HACKELL N. L. BELL AgResearch Ruakura Research Centre Private Bag 3123 Hamilton 3240, New Zealand pip.gerard@agresearch.co.nz Abstract The effects of root herbivory at five densities of Sitona lepidus larvae (overall means between 4 and 333 larvae m -2 ) were assessed over 2 years on newly established perennial ryegrass-white clover swards in a small plot trial. Initial larval establishment in autumn 2003 was positively related to clover content in plots, and there was no significant impact on clover herbage yield in the first year. Nodule damage in winter 2003 increased with larval density, and results suggested an overcompensatory response in nodule production. A 34-35% reduction in clover yield between highest and lowest S. lepidus densities was recorded for both cultivars in the second year, with greatest losses in spring This coincided with reductions in clover root and stolon weights. Plant parasitic nematodes and grass grub larvae were most abundant in the plots with lowest weevil numbers. These results confirm field observations that S. lepidus is a major pest of pastures. Keywords clover; Costelytra zealandica; nematodes; root herbivory; Sitona lepidus INTRODUCTION Since it was first identified in Waikato pastures in 1996 (Barratt et al. 1996), the clover root weevil, Sitona lepidus Gyllenhal (Coleoptera: Curculionidae), has A07005; Online publication date 5 July 2007 Received 12 January 2007; accepted 30 May 2007 spread rapidly throughout the North Island of New Zealand. Monitoring of weevil populations in white clover-ryegrass pastures has shown there are two generations a year in the North Island, with adults emerging in late spring and again in autumn (Gerard et al. 1999). The adults feed on clover foliage, favouring newly germinated seedlings (Hardwick & Harens 2000). Although the adult feeding notches on clover foliage are the most obvious symptom of weevil infestation, the soil-dwelling larvae are the most damaging stage, especially during winter when numbers commonly exceed 400 larvae m" 2 in dairy pastures. The first instar larvae feed on nodules, then as they mature, the lateral roots and finally the nodal roots and stolons (Gerard 2001; Gerard et al. 2004). Assessments of pasture impacts to date have relied on farmer opinion and circumstantial evidence from production data gathered before and after weevil incursion (Eerens et al. 1998; Gerard et al. 2004). These have suggested that the weevil has a severe effect on white clover, Trifolium repens L. (Fabaceae). However, many other factors are known to affect clover persistence and performance within pastures, including climate, pasture management practices, and other root herbivores. The application of insecticides is a simple and effective experimental tool to measure collective root herbivore impacts on pastures (Goldson 1983; Watson et al. 1985; Watson & Mercer 2000). However, soil insecticides are not target-specific and changes in abundance of non-target organisms may contribute to the observed responses. This is of particular relevance in northern New Zealand pastures where clovers are attacked by a complex of root-feeding invertebrates, including grass grub Costelytra zealandica (White) (Coleoptera: Scarabaeidae), whitefringed weevil Naupactus leucoloma (Boheman) (Coleoptera: Curculionidae) and the nematodes Heterodera trifolii Goffart (Tylenchida: Heteroderidae), Meloidogyne hapla Chitwood and M. trifoliophila Bernard & Eisenback (Tylenchida: Meloidogynidae). The plant parasitic nematodes in

3 382 New Zealand Journal of agricultural Research, 2007, Vol. 50 particular have a very significant impact on white clover persistence and production in New Zealand (Watson & Mercer 2000). Obtaining good data on the reductions in seasonal pasture performance caused by S. lepidus was necessary for the assessment of the cost effectiveness of pest management strategies. Because it was difficult to obtain reliable S. lepidus impact data from surveys or on-farm insecticide trials, a small-plot trial was commenced in late 2002 in which S. lepidus larval numbers were manipulated by either the addition of eggs or the exclusion of adults. The objective of the trial was to determine the impact of S. lepidus larvae on clover growth and herbage dry matter production in a mixed perennial ryegrass-white clover sward. METHODS A randomised block experiment was run in six 1 x 10 m cold frame beds at the Ruakura Research centre, Hamilton, New Zealand. each cold frame bed was divided into ten 1 1 m plots using 15 cm deep wooden barriers to prevent movement of roots and larvae outside the plot area, giving a total of 60 plots. The beds were filled with Horotiu silt loam soil, which had been taken from a maize (Zea mays L.) field to ensure clover root pest levels were very low and would not impact on clover establishment. Soil fertility was monitored during the trial and potassium sulphate was applied in late winter-early spring (august, September and October) in both 2003 and 2004 at 60 kg ha 1 month 1 to ensure good clover growth. The Olsen P level averaged 29 mg kg 1 in winter during early summer 2002, perennial ryegrass (Loliumperenne L. cv. 'aries Hd') was sown in six drill rows 15 cm apart at a sowing rate of 2 g m 2 across all plots. To replicate typical farm conditions in Waikato, plots were irrigated as required to ensure good establishment and plant vigour but were otherwise exposed to prevailing climatic conditions. Weeds were controlled during establishment, and those that had potential to take over plots, in particular broad-leaved dock (Rumex obtusifolius L.), giant buttercup (Ranunculus acris L.), creeping mallow (Modiola caroliniana (L.)), and paspalum (Paspalum dilatatum Poir.) were removed periodically during the experiment. although some root disturbance was inevitable, careful hand weeding with a sharp blade rather than herbicide was the method of choice given the diversity and vigour of weeds present. Two contrasting white clover cultivars were used: 'Grasslands Prestige' (a small-leaved early flowering clover) and 'Grasslands kopu' (a large-leaved later flowering clover). Five plots within each cold frame bed were randomly allocated to each cultivar. To prevent S. lepidus entering the plots until autumn, clover plants were established in small pots in S. lepidus-free soil taken from maize paddocks. Nine well-established stolonating plants were transplanted into each plot in an approximately 3 3 pattern in late summer 2003 and supplementary clover seed sown between the ryegrass drill rows at g m 2. Once the clover was established, plots were mown with a rotary garden mower, with catcher attached, to 3-5 cm height depending on growing conditions. To simulate grazing management practices most suited to the contrasting growth habits of the two cultivars, the 'Prestige' plots were normally cut twice as often as 'kopu'. The intervals between cuts varied with pasture growth, with 'kopu' cut usually when it reached a height between cm and 'Prestige' 6-10 cm. The plots of each clover cultivar within a bed were randomly allocated one of five S. lepidus densities (control, low, medium-low, medium-high, and high). The site was closely monitored for any adult feeding damage and during the main periods of adult activity in autumn and spring in 2003 and autumn 2004, the control density was maintained by chemical and temporary physical barriers. Fastac (active ingredient alpha-cypermethrin) was applied at approximately 14-day intervals. This insecticide has quick knockdown activity against adult S. lepidus but is not mobile in soil. Therefore, Fastac applied to dry foliage was highly unlikely to have any direct activity against S. lepidus larvae and other root herbivores present below ground in this trial. each control plot was surrounded completely by a 25 cm high wall of Corflute (hollow fluted plastic board) pushed 2-3 cm into the soil, with an approximately 4 cm band of Stickem insect trapping adhesive around the upper inside surface. The low and medium-low densities were initiated by wild S. lepidus adults that entered the plots from the surrounding landscape, with Fastac used three times in spring and in each autumn (a total of nine occasions) to reduce numbers establishing in the low density plots. The medium-high and high densities were augmented by S. lepidus eggs added throughout the above periods of adult activity. eggs were obtained from S. lepidus adults collected from a ryegrass-white clover pasture close to Hamilton using a modified Homelite HB180V blower/vac and

4 Gerard et al. Impact of Sitona lepidus larvae 383 held in the laboratory in mesh-based tops of twotier cages along with a supply of clover. The eggs dropped through the 0.5 mm mesh and collected in the cage base. approximately 700 and 1400 eggs m 2 were added to each medium-high and high plot respectively each season. This is roughly equivalent to the number of eggs laid by 4-8 fully reproductive female S. lepidus in a month (Markkula 1959). This method was used in preference to caging adults on plots because insect-tight cages prevent the use of a mower, greatly hamper plot maintenance and the collection of plant and insect samples, and create a microclimate that fosters outbreaks of aphids and plant pathogens. Root herbivores Four 7.6 cm diameter soil cores to a depth of approximately 10 cm were taken from each plot to assess numbers of S. lepidus larvae on four occasions: July 2003 (winter), March 2004 (early autumn), September 2004 (early spring), and March 2005 (autumn). The timing of the larval sampling was judged from natural populations in nearby pastures and provided estimates of the peak larval numbers arising from eggs laid over the months following the spring and autumn adult emergence periods. The cores were hand sorted for S. lepidus and other soil-dwelling pasture pests, so that the soil and invertebrates could be returned to the plots from which they had been taken after sorting. The pest larvae present at the sampling dates were of sufficient size that recovery by hand sorting by a skilled operator would not differ statistically from floatation extraction methods. While some mortality would occur during this process, it was considered less disruptive to plot integrity than refilling holes with soil from an outside source. Nematode populations were sampled in late spring (November) Three cores (each 2.5 cm diameter x 10 cm depth) were taken from the high and control plots of both cultivars in a diagonal transect avoiding the outer c. 20 cm of the plot to avoid edge effects. areas previously sampled for insect determinations were also avoided. The soil from each plot was hand crumbled, mixed, and the nematodes extracted from a 100 g subsample by a modified Whitehead and Hemming tray method (Bell & Watson 2001). Total nematode abundance was estimated using a doncaster counting dish. after counting, extracted nematodes were examined fresh at magnification and plant parasites identified to genus. No assessments were made of leaf herbivore density in plots, including S. lepidus adults. Pasture yield and composition Before mowing, a 0.1 m 2 square quadrat of herbage was cut from each plot to the planned mowing height. The herbage was sorted into clover, grass, and weeds, dried and weighed. The weed data were not analysed as periodic hand weeding of some of the larger weed species had been undertaken. The herbage measurements commenced in autumn 2003 and finished in autumn As 'Prestige' plots were cut more often than the 'kopu' plots, the data taken from the intervening harvests were pooled with that collected at the same time as the 'kopu' harvests, to give a total of 17 harvests for analysis. In the first year of the trial, point analyses of all plots were carried out days after mowing using a point quadrat apparatus (Lynch 1966). Ten points were taken in each plot and cover of grass, clover, weeds and bare ground assessed. Once the sward became dense, point analysis was discontinued and pasture composition assessed solely from herbage dissections. Clover material present in the 7.6 cm diameter soil cores taken for larval assessments was washed and separated into root, stolon, and shoot fractions before being dried and weighed. Nodulation and N fixation damaged and active (red leghaemoglobin pigmentation present) nodules were counted for the winter 2003 root samples only. The proportion of clover nitrogen (N) fixed from the atmosphere in the control and high plots was determined in spring 2003, autumn and spring 2004 and autumn 2005 using 15 N isotope dilution (Ledgard et al. 1985). all plots were cut to a sward height of 5 cm and 15 N-enriched ammonium sulphate solution was applied evenly to control and high plots at a rate of 1 kg N ha 1 using a watering can with a rosette attachment, followed by water to wash off any residue on the foliage. a month later, one herbage sample per treated plot was collected, separated into clover and grass, dried, finely ground and analysed by mass spectrometer at Lincoln University. Statistical methods and analysis data were analysed by analysis of variance and linear regression equations were obtained using GenStat, 8th ed. (GenStat committee 2005). The analysis was a factorial randomised block fitting two cultivars and five S. lepidus densities. The cover data and dry matter data were analysed separately for each time and with a repeated measures analysis including all times. clover and grass cover were analysed as a percentage of total plant cover, thus were independent

5 384 New Zealand Journal of agricultural Research, 2007, Vol. 50 of the bare ground variable. The larval density and nematode data were log-transformed to equalise the variance to better meet the normality assumptions of the analysis. Bias-corrected back-transformed means and SEDs are presented with the significance from the log analysis. RESULTS Clover root weevil The planned range of very low to high larval densities was achieved and maintained in the designated plots throughout most of the trial period, as indicated by the larval densities observed at the first three invertebrate sampling dates (Table 1). The mediumlow to high densities of S. lepidus differed significantly from the control density at all sampling times, but the low density did not (Table 1). The number of larvae arising from eggs laid over the summer (March 2004) was smaller than the numbers through autumn and winter (July 2003 and September 2004) (Table 1). Only six S. lepidus larvae were recovered from all plots in autumn (March) 2005 following above-average hot, dry conditions from mid January into March. There was no significant difference in larval densities between cultivars at any of the sampling dates. At the first assessment in July 2003 (winter), plots with good clover cover had better larval establishment than those with low clover. For instance, plots with 50% or greater clover cover averaged 305 ± 81 larvae m 2 compared with 143 ± 26 larvae m 2 for plots with 20% or less clover cover. When all but the control plot larvae numbers were averaged at each point analysis clover cover score, a linear relationship was found between mean numbers of S. lepidus larvae present (y) and percent clover cover (y = 3.9x + 69, R 2 = 0.78, P < 0.01, n = 7). Other root herbivores Whitefringed weevil density was less than 15 m 2 and distributed randomly throughout the trial site Table 1 Mean densities of Sitona lepidus larvae in plots of white clover/perennial ryegrass during the 2003 winter generation (July 2003), summer generation (March 2004) and 2004 winter generation (September 2004). data from 'kopu' and 'Prestige' white clover plots combined., P < S. lepidus density control Low Medium-low Medium-high High Sed 1 Jul Mar Mean larvae m 2 Sep Bias-corrected back-transformed means and SEDs with the significance from the log analysis. Trial mean Table 2 Mean numbers of predominant genera and total plant parasitic nematodes extracted from 100 g soil from control and high Sitona lepidus plots in late spring 2004., Not significant;, P< 0.05;,P< Genus Pratylenchus Heterodera Helicotylenchus Paratrichodorus Rotylenchus Total plant parasites common name Lesion cyst Spiral Stubby root Spiral S. lepidus density control High Sed Bias-corrected back-transformed means and SEDs with the significance from the log analysis.

6 Gerard et al. Impact of Sitona lepidus larvae 385 at all sampling dates. There was no relationship between the density of this weevil and S. lepidus density, plot clover content or cultivar. Grass grub was present during most of the trial, but at the first three sampling dates levels were low, exceeding 25 larvae m 2 in the 'Prestige' control and 'kopu' medium-low rates only in March In March 2005, however, a number of plots had in excess of 200 larvae m 2. Grass grub infestation levels inversely correlated with log 10 numbers of S. lepidus larvae recorded the previous spring (y = -5 4x + 203; R 2 = 0.89; P< 0.05) (Fig. 1). More plant parasitic nematodes were found in the control plots than the high S. lepidus plots (P < 0.01), in particular nematodes in the genera Heterodera (P < 0.05) (Table 2). Meloidogyne (root knot) and Paratylenchus (pin) nematodes were observed in numbers too low to analyse. Pasture yield and composition at sward establishment, the prostrate, densely stolonated 'Prestige' was faster spreading than the erect 'Kopu', with 'Prestige' plots having significantly less bare ground and more clover cover at the early sampling dates (Table 3). Therefore 'Prestige' dry matter (dm) yields exceeded that of 'kopu' for the first four of the 17 herbage yield assessments, although the difference was only significant in late spring (118 versus 76 kg ha 1 respectively since previous harvest, Sed = 13 kg ha 1, P < 0.01) i R 2-54x = g^ S. lepidus Fig. 1 Relationship betweenmeancostelytrazealandica larval densities (±SeM) in early autumn 2005 and mean log Sitona lepidus larval densities in spring Thereafter, 'kopu' yields exceeded those of 'Prestige', which is reflected in the annual clover DM yields in both Year 1 and Year 2 (Table 4). The experimental method accentuated the cultivar difference, as, with a prostrate growth habit, much of the 'Prestige' foliage was below cutting height during the cooler months. The repeated measures analysis of bare ground, clover cover and grass cover variables all had a significant time cultivar interaction. 'kopu' plots had a higher proportion of grass in the plant cover Table 3 Percentage of 'kopu' and 'Prestige' plot surface covered by bare ground, and clover and grass; percentage of plant cover at five times during first year of clover-ryegrass sward establishment., Not significant;, P < 0.05;, P < 0.01;, P < Bare ground 'kopu' 'Prestige' Sed clover % of plant cover 'kopu' 'Prestige' Sed Grass % of plant cover 'kopu' 'Prestige' Sed Winter l Sep Spring % cover Nov dec Summer Feb

7 386 New Zealand Journal of agricultural Research, 2007, Vol. 50 at the first two sampling dates in winter (July) and early spring (September) 2003 (Table 3). The mean annual clover, ryegrass and total pasture DM yields for the five S. lepidus densities for Year 1 and Year 2 are shown in Table 4. during the first year following establishment ( ), there was no significant effect of S. lepidus density on yield. However, in the second year, significant differences in clover yield were found among the S. lepidus densities, with a 34-35% yield reduction between high and control densities for both cultivars. The loss in clover yield was reflected in a reduction of total dm produced in for both cultivars with the medium-high and high 'kopu' plots producing significantly less total DM than the control 'kopu' plots (P < 0.05). Likewise, the medium-low and high 'Prestige' plots produced less than the controls (P < 0.05). The repeated measures analysis of the 2 years had a significant year larval density interaction for 'kopu' clover and total dry matter, but not for 'Prestige' variables. When herbage yields were combined to compare seasonal production, control and high density plots showed greatest differences in clover dm in spring 2004 (Fig. 2). Yield was also significantly lower in high compared to control plots in winter 2004 for both cultivars, and 'Prestige' in autumn Sitona lepidus density had no significant direct effect on ryegrass production in either Year 1 or Year 2. However, there was a significant negative linear relationship between mean clover (y) and ryegrass (x) yields for 'kopu' (y = x + 565, R 2 = 0.85, P < 0.001) for the period. additionally, pooling the 2 years for 'Prestige' gave significant ryegrass (P < 0.05) and total dm (P < 0.05) differences between S. lepidus densities for this cultivar, due to the high yield in both years at the low density (Table 4). The total dm yields from 'Prestige' and 'kopu' plots showed similar response patterns to S. lepidus density in the second year. Only at the spring (September) 2004 assessment was there any statistically significant impact on root and stolon dm (Table 5). Increasing S. lepidus larval numbers were negatively related to the amount of clover roots present in 'kopu' plots (linear trend, P < 0.05). The medium-low density (214 larvae m 2 ) plots had the lowest stolon and root weights when data were pooled for cultivars, and root dm m 2 in the medium-low density treatment was significantly lower than the control and low plots for 'Prestige'(Table 5). Nodulation and N fixation although 'kopu' had fewer but larger nodules than 'Prestige' (overall means 7856 versus nodules m 2, P < 0.001), the proportion of nodules damaged in winter 2003 was similar for both cultivars and the pooled results are presented in Fig. 3. Table 4 effect of S. lepidus density on mean yearly clover, grass and total pasture yield including weeds (kg dm ha 1 ) from 'Kopu' and "Prestige' clover-ryegrass swards during , Not significant;, P < 0.05;,P<0.01. 'kopu' clover Yr1 clover Yr 2 Ryegrass Yr 1 Ryegrass Yr 2 Total dm Yr1 Total dm Yr 2 'Prestige' clover Yr1 clover Yr 2 Ryegrass Yr 1 Ryegrass Yr 2 Total dm Yr 1 Total dm Yr 2 control Low Sitona lepidus density Medium-low Medium-high Results are from separate analysis of variance analyses for each cultivar High Sed P

8 Gerard et al. Impact of Sitona lepidus larvae 387 Fig. 2 effect of control and high Sitona lepidus (320 larvae m 2 ) densities on seasonal white clover yield (kg dm ha 1 ) of A, 'kopu' and B, 'Prestige' clover-ryegrass swards Vertical bars are Seds. Control High Win Spr Sum Ant Win Spr Sum See»fi (autumn summer 2005) The mean number of damaged nodules increased with S. lepidus larval densities present at that time (y = 7.071x + 421, R 2 = 0.97, P< 0.01), withmediumlow, medium-high and high plots having a significantly higher proportion of damaged nodules than low and control plots (P < 0.001). However, total nodules also increased at the higher densities, with medium-high having significantly more nodules than control and low plots (P < 0.05), and high plots having significantly more nodules than control, low and medium-low plots (P < 0.05). In both Year 1 and Year 2, the percentage of N fixed from the atmosphere was higher in spring (November) than in autumn (April) (Table 6, P < 0.001).

9 388 New Zealand Journal of agricultural Research, 2007, Vol z X Larvae m Fig. 3 Relationship between mean total and damaged white clover nodules (±SeM) recovered and mean Sitona lepidus larval density in July There was no difference in N fixation between cultivars except in autumn 2005 when 'kopu' averaged 67% N fixed compared to 59% for 'Prestige' (P < 0.05). Although percentage N fixed in high S. lepidus density plots in autumn 2004 averaged 19% lower than in the controls, this was not statistically significant using analysis of variance (Table 6). However, if the autumn 2004 control and high plot data for each cultivar within each cold frame bed are paired, the difference becomes significant (paired t-test, P < 0.05). Using plot clover content at time of N fixation measurement to calculate N fixed ha 1, control plots fixed 59% more N ha 1 in autumn 2004 (P < 0.05) and 83% more in the following spring (P < 0.01) than the high plots (Table 6). Table 5 effect of winter generation Sitona lepidus larval density on mean white clover stolon and root weights/plot (g dm m 2 ) in 'kopu' and 'Prestige' clover-ryegrass swards in spring (September) 2004., Not significant;, P < 0.05;, P < 'kopu' Stolon Root 'Prestige' Stolon Root Pooled Stolon Root control Low S. lepidus density Medium-low Medium-high High Sed P Table 6 effect of control (4 larvae m 2 ) and high Sitona lepidus (333 larvae m 2 ) densities on percentage and amount of N fixed by white clover in clover-ryegrass swards at assessments in autumn and spring , Not significant;, P < 0.05;, P < %N fixed Spring 2003 autumn 2004 Spring 2004 autumn 2005 kg N fixed ha 1 Spring 2003 autumn 2004 Spring 2004 autumn 2005 control S. lepidus density High Sed P

10 Gerard et al. Impact of Sitona lepidus larvae 389 DISCUSSION The positive relationship between the levels of S. lepidus larvae and percentage of clover in plots soon after establishment demonstrated the dependency of the weevil on its host plant. although not analysed in the context of this paper, this density dependence and resultant feedback on clover indices masked initial impacts of S. lepidus root and nodule herbivory on clover yield in the first year. The larval populations were typical of those found in New Zealand pastures in the post-invasion equilibrium phase (Gerard unpubl. data). as the clover content was higher than most pastures infested with S. lepidus (Gerard et al. 2004), the pressure on clovers in this trial was less than in pastures with equivalent S. lepidus densities. Nevertheless, in the second year the pressure of prolonged larval damage reduced clover annual dm yields by around 34%. While the losses were most apparent in late winter and spring, the causative damage would have commenced in the previous autumn when newly hatched larvae attacked the nodules. The very close relationship between number of damaged nodules and S. lepidus density in winter (July) 2003 (Fig. 3) indicated that the nodule damage observed was herbivory by first instar S. lepidus larvae. although a quadratic curve was fitted to the total nodule data in Fig. 3, a sigmoid curve suggesting a possible overcompensatory response in nodule production above a threshold of 200 larvae m 2 may be more appropriate. Quinn & Hall (1992) observed an overcompensatory response in lucerne (Medicago sativa L. (Fabaceae)) to nodule herbivory by first instar S. hispidulus larvae, with overcompensation in number of nodules at day 10 of the laboratory experiment, but exact compensation by day 18. In our trial, eggs had been added to the high plots from mid april until late May 2003 and therefore the exposure to newly hatched S. lepidus was far more prolonged than in the Quinn & Hall (1992) laboratory experiment. as only a single nodule assessment was made and nodule dm was not assessed, it was not possible to determine if nodule size decreased with increasing nodule number, or to verify whether compensatory responses in nodule number were occurring. However, either of these compensatory responses in nodule production would have created an increased sink for N in the root system, caused a shift in the c:n balance throughout the plant, and in turn reduced herbage dm yield as shown in the laboratory by Murray et al. (1996). Lucerne compensated for the loss of nodules through larval herbivory by the related species Sitona discoideus Gyllenhal until foliar N levels dropped below 4.7%, at which level regeneration of nodules required more N than was being fixed and herbage yield abruptly decreased (Goldson et al. 1988). a similar response was observed in pure swards of'kopu' and 'Prestige' white clover with S. lepidus larvae associated with decreased percent foliar N levels in both cultivars in spring, and subsequent reductions in herbage DM yield levels in 'kopu' (Gerard 2002). In late winter and early spring most larvae are in the final instar and therefore feeding on stolons and nodal roots (Gerard et al. 2004). as with S. hispidulus (Powell & campbell 1983), this causes general disruption to the transport system through damage to the vascular tissue. However, this damage also coincides with the formation of new nodal roots and stolons (Brock & Hay 1996). Therefore, the plant is able to regenerate new tissues, albeit by depleting carbohydrate reserves (Murray et al. 1996). This diversion of carbohydrates and N reserves to the nodules, stolons and roots may well explain why, although the primary damage to the root system resulted in only minor reductions to stolon and root DM (Table 5) and percentage N fixed (Table 6), the secondary effects on foliar production were much more significant (Fig. 2). Overall, the two cultivars responded similarly to attack by S. lepidus larvae. Herbage dm yield losses were greatest in winter and spring, and both cultivars showed some compensatory increase in yield when S. lepidus pressure disappeared in late summer 2005 compared to the decline observed in the controls (Fig. 2) that typically occurs in cloverryegrass swards over summer (Ledgard et al. 1990). 'Prestige' appeared to suffer significant losses in DM production earlier than 'kopu', but recovered faster, probably because it has higher stolon density and more nodal points than 'kopu' from which to grow new roots (Caradus & Woodfield 1997). clover root herbivory by Sitona hispidulus F. has been shown to increase dm yields in companion ryegrass in the laboratory by accelerating the transfer of N (Murray & Hatch 1994). It is possible that the high ryegrass yield from the low 'Prestige' plots in both years is evidence of this, and that the benefits disappear at higher levels of herbivory. However, the 'kopu' dm results indicate S. lepidus has a more direct influence on the competitive interaction between clovers and grasses. decreasing S. lepidus root herbivory resulted in increasing clover dominance in 'kopu' plots with the large-leafed clover shading and out-competing the ryegrass in Year 2 (Table 4).

11 390 New Zealand Journal of agricultural Research, 2007, Vol. 50 The presence of both C. zealandica and plant parasitic nematodes in greatest abundance in the control S. lepidus plots confirms that the insecticide applications had no direct impact on other major clover root herbivores. The difference in nematode levels between the control and high S. lepidus rates probably reflects the differing quantity of roots. The inverse relationship between C. zealandica abundance and spring S. lepidus larval populations was unexpected and was most likely a response to the variation in white clover content with S. lepidus density. even though C. zealandica has a relatively diverse host range, the pasture white clover:ryegrass ratio is known to have a marked effect on early instar C. zealandica larval establishment, as, in contrast to third instars, first instar larvae survival is very poor on ryegrass (kain & atkinson 1977). Therefore, C. zealandica first instar establishment may have been hampered in plots where clover root availability was impaired by S. lepidus herbivory in comparison to control plots where little or no feeding activity was taking place. apart from clover abundance, other possible explanations include adult C. zealandica oviposition preferences and interspecies competition for nodules, behaviours not previously associated with C. zealandica. a number of studies have found that ovipositing C. zealandica females show little preference between pasture species or height (kain & atkinson 1977 and references therein). as C. zealandica and plant parasitic nematodes population levels were the inverse of the S. lepidus rates, it is probable that the differences in clover dm production between the control and high S. lepidus rates were less than if these pests were absent or randomly dispersed across the trial site. However, this underestimation is only of theoretical interest since parasitic nematodes, at least, are ubiquitous in pastures. It is possible that the low root and stolon dm levels in medium-low S. lepidus density at the spring (September) 2004 assessment arose from the combined pressure from nematodes, S. lepidus, and competition from companion ryegrass. This trial was designed to simulate natural pasture conditions so that the damage measurements could be related to on-farm S. lepidus populations. However, small-plot trials in the absence of animal grazing have limitations, the main one relevant to this experiment being that the swards tend to be clover dominant (Lynch 1966). Ruminants tend to graze selectively, showing a preference for white clover (Cosgrove et al. 1996). The sward was even, lacking the typical patchiness of grazed pastures, and the mowing regime ensured that the sward never became rank and that the stolons were never exposed to high soil surface temperatures in summer. Therefore, the clovers in this trial were under very little stress compared to those in pastures. The resulting dominance of clover was apparent within the first year (Table 3), and was particularly marked in the second year in the low S. lepidus density 'kopu' plots where the large-leafed clover shaded and outcompeted the ryegrass (Table 4). apart from the treatments imposed, the management of the small plots would have had little impact on S. lepidus biology and ecology compared to that in pastures. Both mowing and grazing would expose eggs and adults to desiccation in a similar manner. S. lepidus adults feed in short bursts followed by long periods hidden in the base of the sward (Gerard & Hackell 2005), thus avoiding both animal ingestion and removal by mowing. as with adult argentine stem weevil (Listronotus bonariensis (kuschel)) populations in Waikato pastures (Barker & Addison 1995), it is unlikely stock treading has any marked direct impact on populations of the robust adults. Soil compaction and pugging under high intensity grazing regimes would impact on both plant and soil invertebrate populations, but those interactions were beyond the scope of this study. egg predation by ants was considered the main trial artefact to interfere with S. lepidus populations. ant bait was laid to eliminate the multitude of nests associated with the cold frame beds, but reinvasion was continuous in the warmer months. In conclusion, root herbivory by S. lepidus larvae at densities typical of those in Waikato pastures significantly reduced herbage yields of white clover grown in a clover-ryegrass sward. The trial results confirmed New Zealand farmer observations (Eerens et al. 1998) and a previous United kingdom field study (Mowat & Shakeel 1988) that attributed yield losses to S. lepidus larval damage. The yield loss was greatest in spring, which coincides with grazing animals in high nutrient demand, such as rapidly growing young animals and lactating cows and ewes. With white clover being considered the best quality component of grazed pastures because of its high nutritive and feeding value (caradus et al. 1996), it is evident why New Zealand farmers regard S. lepidus as a major pest species. ACKNOWLEDGMENTS We thank Paul Addison for establishing the trial, Derrick Wilson and Tina Eden for assistance with assessments and

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