Root feeding organisms can consume significant amounts of plant material, and their control is a considerable expense in many agricultural systems, including some grasslands used for pasture and forage production.  However, despite the potential of below ground herbivory as a key ecological interaction in grasslands, little research has been done to examine the effects of root feeding on soil biodiversity and processes.  Recent work has demonstrated that below ground herbivory can result in changes in patterns of rhizodeposition, root turnover, and root distribution that can impact on rhizosphere carbon flow and soil microbial diversity and functioning.      This experiment aims to evaluate the indirect effects of invertebrate root feeders on soil microbial communities.  Root feeding organisms include invertebrate root herbivores such as plant parasitic nematodes and the larval stages of many insects.       This experiment will test the hypotheses that: (1) Root feeders enhance and accelerate the transfer of plant-derived carbon into and through the soil microbial biomass. (2) Root feeding affects soil microbial diversity and activity. (3) Root feeding affects plant biomass and the architecture of root systems. (4) Root feeders affect the colonisation of roots by arbuscular mycorrhizae. To test these hypotheses, we propose to initiate a pot experiment, using monocultures of Agrostis capillaris. A. capillaris is the dominant grass species in many upland grasslands, including the Sourhope NERC Soil Biodiversity Thematic Programme site in the Scottish Borders.  A. capillaris will be subjected to root feeder treatments in order to evaluate the direct and indirect effects of these organisms in simple grass-soil systems.  Pots (15cm diameter, 15cm deep) will be filled with soil collected from the Sourhope field site that has been sieved (6 mm) to remove stones and plant material.  Soil was collected from the Fasset Hill site (unimproved grassland, class U4A) at Sourhope.  The turf (0-4inches) was scraped off and the soil collected from underneath (4-12 inches depth).  Soil will be defaunated by a heat treatment (80C, 24 hours), a freezing treatment (-20C, 24 hours), and another subsequent heat treatment (80C, 24 hours) in order to eliminate communities of root feeders that are present.  Pots containing defaunated soil will be inoculated with 100 ml of a muddy soil wash made from filtering a soil suspension (250 g soil/1000ml water; shaken 15 minutes) through a 53um sieve, in order to ensure that microbial inoculum is available.  Application of the soil wash will occur shortly after germination of seeds. Subsamples of the defaunated soil (4) will be tested to ensure elimination of insect larvae and nematodes.  The same soils will also be subjected to microbial analyses for comparison with non-defaunated, control soil.  Defaunation is likely to have a considerable impact on soil carbon, structure, and micro-organisms.  However, there will be a long equilibration period before the treatments are applied, while the plants are growing.  During this period, it is likely that the defaunated soils will stabilize.  The experiment will be contained in a Conviron growth room at 20C, with a 14 h photoperiod during the pre-treatment period of plant growth.  Nutrients will be supplied to plants if there appear to be any deficiencies.  Water will be supplied as needed.  During the treatment period, water and nutrient supply will be measured to ensure equal application to all pots. At the time that treatments are applied, the growth room temperature will be lowered to 12C during the light part of the cycle and 10C during the dark part of the cycle. After 1 week, temperature will be raised to 15C during the light and 10C during the dark.  Soil temperature will be monitored throughout the experiment.  Each pot will be sown with the same mass of Agrostis capillaris seeds at reseed densities (25g/m2, 0.44g/pot).  Pots will be will be watered as needed to maintain field capacity and nutrient solution (full-strength Hoagland's) applied every other week after treatments are initiated.  Plants will be clipped (to 5 cm) as needed to prevent seeding.  Plants will grow for approximately eight weeks, in order to establish soil structure, mycorrhizal associations, and a turning-over root system.  At the conclusion of the growth period, invertebrates will be introduced to half of the pots. Treatments (All defaunated, except 1, 6, 7 & 12): 1) Control (non-defaunated soil) 2) Control  3) Tipulid 4) Nematode 5) Nematode + Tipulid 6) Tipula + Nematode (non-defaunated soil)  7) Control (non-defaunated soil) LABELLED 8) Control LABELLED 9) Tipulid LABELLED 10) Nematode LABELLED 11) Nematode + Tipulid LABELLED 12) Tipula + Nematode (non-defaunated soil) LABELLED All 12 treatments are replicated 8 times (except the nondefaunated soil treatments which are replicated 6 times), and arrayed in 8 randomized complete blocks.  Tipulid density will be 8/pot.  Tipulids will be collected by hand from the MLURI field site and maintained in the laboratory (6C) until used for the experiment.  Nematodes will be collected from Agrostis turfs collected from an IGER-Aberystwyth field site.  Nematode solutions will be condensed, overall density and diversity estimated, and then portions will be added to the pots at field densities on two separate dates.  Herbivory treatments will run for approximately six weeks.  Towards the end of the experiment, a 13C pulse label will be applied to half of the pots.  At this time, pots will be moved outside, to the MLURI 'cage' area, for the remainder of the experiment.  The pots will be subsequently sampled to determine 13C concentration in living plant material (roots and shoots) and the soil microbial biomass.  Root feeders (tipulids, etc) may also be analysed for label content.  Samples will be taken for 13C analysis after application of the pulse.  At harvest of the entire experiment, the following analysis will be performed: Plant biomass and C:N (root, shoot) (MLURI) Microbial biomass(MLURI) Biolog; plate counts (MLURI) PLFA (MLURI) Tipulid number (MLURI) Nematodes: Root infection (IGER-Aber) Root architecture, proportion living/dead (MLURI-LD/JR) Arbuscular mycorrhizal colonisation of roots (% RLC) (RH) Experiment Schedule:   TASK Date completed Potting soil and soil defaunation (oven and freezer) 12.2.01 thru 15.2.01 Pots wetted and seeded 15.2.01 Seeds germinated 20.2.01 Microbe wash added 27.2.01 Defoliation (5cm height) 13.3.01 Defoliation (5cm height) 23.3.01 Defoliation (5cm height) 6.4.01 Tipulid Collection (MLURI) week of 17.4.01 Moved to new growth room 23.4.01 Defoliation 24.4.01 Started application of full strength Hoaglands weekly (100ml); water application standardized between plants 27.4.01 Application of treatments:  Nematodes 3 May  Application of treatments:  Tipulids 2 & 6 May Movement of pots from growth room to cage 29.5.01 Application of 13C pulse  31 May Sampling of labelled cores 11 June   Sampling of labelled cores 25 June  Destructive Harvest 16 July