The pulse labelling method (PLM) is based on the artificial labelling of plants. The technique can be roughly divided into three methods based on the levels of 13C enrichment achieved in the plant-soil system: natural abundance (Heim and Schmidt 2007; Klumpp et al. 2007; Thornton et al. 2004), near natural abundance (50-500%) (Evershed et al. 2006; Leake et al. 2006) or (highly) enriched >500% (Zak and Kling 2006). Shoots are exposed to CO2 in an atmosphere labelled with 14C, 13C or C. The shoots assimilate the labelled C and translocate a part of it into the soil. This C is incorporated into the root tissue, exuded as high and low-molecular-weight organic substances, sloughed as cell tissues by root elongation and released as CO2 derived from root respiration. Hence, the entire labelled C later found in all soil pools or evolved as CO2 from the soil is plant-derived. This enables the C input by plants in soil to be calculated on the background of soil organic C, which remains unlabelled. The calculations are similar to those used for natural isotope labelling outlined below (the third method). The fractional input (F*) of C from the new 13C natural source in the existing soil C pool (or constituents) can be estimated using a linear mixing model as follows:
d source — d initial where F* is the proportion of new C present in soil, d source is the d13C or atom% 13C of the source C applied to soil, and d final and d initial are the initial and final d13C or atom% 13C of soil C pool at the beginning and end of the experimental period.
Near natural or high enrichment labelled approaches generally use artificially labelled plant materials (Leake et al. 2006) or commercially available 13C-labelled substrates (Evershed et al. 2006; Zak and Kling 2006) to trace C flows. Due to high costs to produce labelled plant material or buy expensive 13C-labelled compounds, most of these approaches are laboratory based (e.g., Evershed et al. 2006) or rely on small field experiments (e.g., Leake et al. 2006; Zak and Kling 2006). Experiments using artificial labelling methods are generally conducted for short periods of time (i.e., weeks to months), hence they generally only completely label those soil components which have a relatively high turnover rate (e.g., soil microbial community and water soluble carbon).
In the case of pulse labelling, the shoots assimilate the labelled CO2 for only a short period, even only once during the whole plant growth. In continuous labelling technique, the plants assimilate labelled CO2 over a long period. Many different experimental systems for pulse and continuous labelling of plants have been described (e.g., Sauerbeck and Johnen 1976; Whipps and Lynch 1983; Shepherd and Davies 1993; Cheng et al. 1993; Jenkinson et al. 1999; Swinnen et al. 1994). The results obtained by pulse labelling correspond to the relative distribution of assimilated C at the moment of labelling and do not reflect the distribution of total unlabelled C in different plant parts. The total amount of C assimilated by the plant is unknown and can only be roughly calculated.
The most important limitation of pulse labelling is that the results of C allocation observed at a specific time or growth stage cannot be directly transferred to the whole growth period. However, a series of labelling pulses applied at regular intervals during plant growth have been found to provide a reasonable estimate of cumulative belowground C input (Jenkinson et al. 1999; Swinnen et al. 1994; Kuzyakov and Schneckenberger 2004).
In the case of continuous labelling, the total amount of assimilated C is known. In addition, the distribution of labelled C corresponds to the distribution of total C, as long as the labelling is applied from first leaf emergence to harvest time (the specific 14C activity or 13C abundance is equal in all plant parts). Therefore, CLM is particularly appropriate for estimating the amount of total C transferred by plants into soil and belowground pools during the all labelling period (Meharg 1994), and is also useful to distinguish root-derived from SOM-derived CO2 (Johnen and Sauerbeck 1977; Whipps 1990). Continuous labelling requires special equipment to expose plants over a long period to 14CO2 with constant 14C specific activity, or to 13CO2 with 13C constant enrichment. In addition, the air temperature and moisture conditions must be controlled inside the labelling chamber.
For both pulse and CLMs, radioactive 14C has been used in most studies. This preferential use of 14C is based on higher sensitivity, lower purchasing and analysis costs, and easier sample preparation than for 13C or 11C. Since 11C has a short halflife (20.4 min), only 14C and 13C are appropriate for continuous labelling.
Unlike in traditional methods, in tracer techniques, the amount of tracer that enters the system is known exactly and it is possible to exactly calculate the balance of C in the atmosphere-plant-soil system, as well as the losses from the system. Traditional methods are less accurate and can only be used to calculate the distribution of C among different C pools.
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