The objective of this experiment is to determine what quantitative and qualitative changes occur in root exudation and microbial communities when insect larvae are feeding on the root systems of grass.  Using Microcosms, we will subject Agrostis capillaris (bentgrass) plants to grazing by Tipula paludosa larvae and collect the exudate solution for chemical analysis.  Exudate solution will also be applied to Sourhope soils in receptor microcosms and the soils analyzed to determine changes in microbial community structure.

It is anticipated that the data from this experiment will be directly comparable to repeat experiments using clover and ryegrass, as well as combinations of the plant species.

Experimental Plan:

Agrostis seeds will be surface sterilized using the para-acetic acid technique and germinated on sand in petri plates.  After 6 d of growth, seeds will be transferred to 12 microcosms (6 seedlings/microcosm) and allowed to grow for 8 weeks, to produce a sward-like appearance and developed root system.  Microcosms will be continually supplied with 1mmol N - nutrient solution throughout the experiment.

Eggs obtained from T. paludosa will be hatched and allowed to develop on a diet of dried grass and clover.  Larvae will be transferred into half of the microcosms (4 larvae/microcosm).

16  microcosms will be established and the treatments randomly allocated:

Larvae treatment (6)
No larvae (6)
Non-plant controls (4)

Exudate solution will be collected 3 times weekly over two weeks (M-W-F). Exudate will be
collected with a wash-through system.  Any nutrient solution in the exudate collection chamber
before the wash is applied will be drawn off all microcosms, taking care not to draw any solution from the sand (slower collection rate).  These exudates will be discarded.  200 ml of sterile 1mmol N nutrient solution will be injected into each microcosm and allowed to drip out for 5 minutes.  Then, the exudate will be collected from the bottom chamber, taking care to recover as much as possible. 

After collection, the exudate solution volume will be split.  One half of the solution will be analyzed for total organic carbon, protein, phenolic acids, amino acids, and free carbohydrates.  A 100 ul aliquot of this solution will also be plated at the beginning and end of the experiment  for total culturable bacteria and fungi, in order estimate the degree of contamination introduced by the larvae. 

The other half of the collected exudate solution (100 ml) will be freeze-dried to concentrate the solution and to eliminate contaminating microbes.  The solution will be reconstituted with 60 ml sterile 1 mmol N nutrient solution and then sent via a pump mechanism to drip into receptor microcosms containing 100 g fresh Sourhope soil at a bulk density of 0.75g/cm3.  The exudates will be supplied at a rate of 18.5 ml/d.  This is to ensure that the soils are kept at a moisture level near field conditions.  Temperature will be maintained at a constant 15C.  Control soils to which 1mmol N nutrient solution is applied (at the same rate as the exudates) will also be sampled.  There will be 18 receptor soil microcosms in total - 6 for larvae exudates, 6 for non-larvae, and 6 nutrient solution controls.  An additional 6 soils will be stored in bags at 15C for the
duration to serve as controls for solution application.  At the conclusion of the experiment, these
soils (24) will be sampled and the microbial communities analyzed (PLFA, Biolog, plating,
biomass, respiration). 

Microcosms will be destructively sampled at the end of the experiment.  Plant tiller and root axes numbers will be determined. Shoot, root, and larval biomass and C:N ratio will be determined.    Root architecture will be assessed. 

TENTATIVE SCHEDULE: