Objective:  The objective of this experiment is to determine how carbon flow from plants into microbial biomass (via root exudates) is changed by grazing.  We will determine the relative quantity and rates of transfer of rhizosphere carbon to soil fungi and bacterial groups.  We hypothesize that the transfer of label will be specific to certain microbial groups and that the transfer will be accelerated with clipping. 

Methodology:  Plots in the field will be labeled in the field with 13C and sampled following clipping.  Our experiment will utilize technology that allows the separation for isotopic analysis of microbial lipid components derived from soils by the PLFA procedure (Gas chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS) (sensu Boschker et al. 1998).  This technique will allow us to determine which groups of microorganisms respond to changes in root exudation caused by defoliation. 

Justification:  The use of a stable isotope (13C) to label plants and soils in the field, combined with analyses of specific microbial groups for incorporation of this label will allow us to take a unique look at rhizosphere dynamics.  While laboratory investigations using 14C labeling techniques have been successful at elucidating the rates of transfer of photosynthetically-fixed carbon into the rhizosphere, little work has been done under field conditions, and no work has been done that separates the microbial response into functional groups at the scale we propose. 

Experimental Plan: 

On 24.8.99 field plots will be established at the Sourhope NERC Soil Biodiversity field site in the 'play area'. Twelve chambers (400-mm diameter) will set up for application of the label.  50 atom % 13C labeled CO2 will be used for the pulse and applied using SID (for details of application rate, etc, see Nick Ostle's proposal).

Half of the chambers will be subject to a clipping treatment to simulate grazing after 2 days of pulsing (on 26.8.99).  Soil samples (20-cm depth) will be collected 24 h following clipping on 27.8.99.

Subsamples of 4mm sieved soil (1.5g) will be taken for PLFA analysis, and will be frozen immediately to prevent further transfer of label throughout the soil.  These samples will be kept frozen (-20?C) until the results are obtained from preliminary analyses to determine if label was incorporated in the bulk soil.  If label incorporation is successful, PLFAs will be extracted from the samples, derivitized, and subject to GC-C-IRMS to determine the concentration of 13C in the fungal fraction and in the fractions characteristic of various bacterial groups.  There will be 18 samples in total to be analyzed by GC-C-IRMS, 6 labelled uncut, 6 labelled cut, & 6 unlabelled uncut. 

Additional subsamples (10g) will be collected, kept cool, and will be analyzed for the community level physiological profiles (CLPPs, e.g., Biolog technique) of the soil microbial communities.  Soil microbial biomass will be determined (chloroform fumigation extraction) as well as standard plate counts of soil microbes (total, fungi, pseudomonads).  Soil moisture content will be determined gravimetrically (10g). 

References:

Bardgett RD, Wardle DA, Yeates GW.  1998.  Linking above-ground and below-ground interactions:  How plant responses to foliar herbivory influence soil organisms.  Soil Biology & Biochemistry.  30:1867-1878.

Boschker HTS, Nold SC, Wellsbury P, Bos D, de Graaf W, Pel R, Parkes RJ, Cappenberg TE.  1998.  Direct linking of microbial populations to specific biogeochemical processes by 13C-labelling of biomarkers.  Nature. 392:801-805.

Grayston SJ, Campbell CD, Bardgett RD, Mawdsley JL, Clegg CD, Ritz K, Griffiths BS, & Elston PJ.  1999a.  Assessing shifts in microbial community structure across a range of grasslands of different management intensity using CLPP, PLFA and community DNA techniques.  Applied & Environmental Microbiology.  (submitted) 

Grayston SJ, Griffith GS, Mawdsley JL, Campbell CD, & Bardgett RD.  1999b.  Accounting for variability in soil microbial communities of temperate upland grassland ecosystems.  Soil Biology & Biochemistry.  (submitted)

Grayston SJ, Murray PJ, Reid EJ, MacDougall R, Ord BG, Gill E.  1999c.  Effect of shoot defoliation on carbon flow and soil microbial communities in the rhizosphere.  Abstracts of the British Soil Science Society 1999 Autumn Conference, Edinburgh. 

Hodge A, Grayston SJ, Ord BG.  1996.  A novel method for characterisation and quantification of plant root exudates.  Plant and Soil.  184:97-104.

Hodge A, Paterson E, Grayston SJ, Campbell CD, Ord BG, Killham K.  1998.  Characterisation and microbial utlisation of exudate material from the rhizosphere of Lolium perenne grown under CO2 enrichment.  Soil Biology & Biochemistry.  30:1033-43.

Williams BL, Grayston SJ, Reid EJ.  1999. Impact of artifical sheeps' urine on the microbial biomass, activity and community structure in two pastures in the Scottish uplands.  Soil Biology & Biochemistry. (submitted)