IMPACT OF INVERTEBRATE ROOT HERBIVORY ON N FLUXES IN WHITE CLOVER - P.J. Murray^[33], D.J. Hatch^[34] and J.B. Cliquet^[35]

SUMMARY

An experiment was carried out to determine the impact of root herbivory on clover plants growing in a set of micro-lysimeters in terms of N fixation and root exudation. The results showed that root feeding by invertebrates significantly reduced N fixation over a 27 day period and caused changes in both the quantity and pattern of root exudation.

Keywords: root herbivory, white clover, exudates.

INTRODUCTION

White clover is by far the most important legume in British agriculture. However, it often fails to thrive. It has become apparent over recent years that a major reason for this lack of persistence is the impact of pests and diseases. Some of the main pests of white clover are weevils of the genus Sitona. Sitona larvae are rhizophagous, feeding initially on the nodules and as the larvae develop, on progressively larger roots. The work reported here describes an experiment to determine the impact of root feeding, by Sitona, on N fixation and efflux of N from the clover roots. N fixation was determined in plants grown on a ¹⁵N-depleted source of N: the plants were subsequently enriched in 15N by acquisition of atmospheric N₂ at natural abundance (0.3663 atom% ¹⁵N). The system allowed collection of root exudates which were analysed for inorganic forms of N and amino acids.

MATERIALS AND METHODS

Seeds of white clover (cv. Donna) were germinated in sand. After 15 days the seedlings were transferred to a hydroponic system, described by Hatch and Murray (1994). The culture solution utilized ¹⁴NH₄ ¹⁴NO₃ (99.99 atom% ¹⁴N) as the N-source. When the seedlings had been in the culture troughs for 20 days an inoculum of Rhizobia was added to the nutrient solution. After a further three days growth, the plants were removed from the culture troughs, washed carefully in demineralized water to remove any traces of the culture solution, and transferred to a series of micro-lysimeters. These were fabricated from the barrels of 50 ml syringes. Each barrel was filled with washed silver sand to the 40 ml mark. A depression was made in the sand for the clover roots and then backfilled with dry sand. The plants were individually fed with an N-free nutrient solution (Murray and Hatch, 1994) by means of a peristaltic pump. Each plant was supplied from a separate channel of the pump to ensure that the delivery of nutrient solution was uniform (1.5ml h^-1). Root exudates were sampled automatically during the experimental period, also by peristaltic pumps. These pumps were programmed to operate for eight sampling periods per day to take an integrated sample over 24 hours.

Neonatal weevil larvae were added to 12 young clover plants, with a further 12 plants used as controls. At either 18 or 27 days after infestation with larvae, the plants were harvested to by removing them from the micro-lysimeters and the roots were washed gently in MgSO₄ remove any larvae. The plants were divided into shoots and roots, dried at 80°C for 24 hours for dry matter determination and finely ground using a modified ball-mill. The roots and shoots were analysed for %N and ¹⁵N atom % using an automated N analyser linked to a mass spectrometer. As the samples were anticipated to be below natural abundance, sub-samples were run initially through the N-analyser to determine the N-content of individual plants. This enabled the appropriate weight of samples containing 100 mg N for atom% determination: the optimum amount for the mass spectrometer efficiency. Sub-samples of root exudates were analysed for amino acid and mineral N contents. All data were analysed using ANOVA.

RESULTS

The results showed that the presence of weevil larvae feeding on the nodules significantly (P<0.001) reduced the plant biomass, %N content and number of nodules over the experimental period (Table 1).

The total number of root nodules present on the plants was indicative of the N fixation of the plants. Table 1 also shows the increase in the ¹⁵N atom% of the plants over the experimental period attributable to N fixation. From these figures it appears that approximately 90 percent of the N in the control plants was derived from the atmosphere by day 27, as compared to only 29 percent of the N in the weevil infested plants.

There were no significant differences in exudation of mineral N from the plant roots. No nitrate was detected in any of the exudates. Ammonium levels in the exudates were low in the 18 days samples (17.7 and 11.7 ppb, +weevils and controls, respectively). After 27 days, the ammonium levels in the exudates had risen with higher concentrations in exudates from the infested plants (401 ppb) than in the controls (162 ppb). After 18 days, the amino acid content of the exudates was greater from the weevil-infested plants (9.2 nM) than from the control plants (5.8 nM). However, by day 27 there was 21 nM of amino acids in the exudates from both treatments. Figure 1 shows the relative proportions of the more common amino acids in the exudates sampled 27 days after infestation.

Table 1. Dry Matter (DM), %N content and number of nodules and atom % ¹⁵N ratios of clover plants infested by weevil larvae (+Weevils) or Control plants at 18, or 27 days after the introduction of larvae.

Figure 1. Relative proportions of the main amino acids found in exudates from roots of plants infested with weevil larvae, or controls.

DISCUSSION

This work showed that root herbivory can have a large impact on the growth, yield and nutrient status of the plant and can also alter the composition of root exudates. This may have further implications for the soil microflora in terms of both activity and diversity. Recent work (Cliquet, et al., 1997) demonstrated active uptake of amino acids from the rhizosphere by grass plants. The release of legume N to the soil via root herbivory is a hitherto under-researched component of the natural cycling of N.

ACKNOWLEDGEMENT

This work was funded by the Biotechnology and Biological Sciences Research Council and the University of Caen.

REFERENCES

Cliquet, J.B., Murray, P.J. & Boucaud, J. 1997. Effect of the arbuscular mycorrhizal fungus Glomus fasciculatumon the uptake of amino nitrogen by Lolium Perenne, New Phytologist. 137: 345–349.

Hatch, D.J. & Murray, P.J. 1994. Transfer of nitrogen from damaged roots of white clover (Trifolium repensL.) to closely associated roots of intact perennial ryegrass (Lolium perenne L.). Plant and Soil. 166: 181–185.