Symbiotic Fungi: Principles and Practice (Soil Biology)

17 15 N Enrichment Methods to Quantify Two-Way 287
inorganic chemicals or organic compounds (up to 99.90 atom 15 N, %) is applied to
plant tissues, growth media or soils, and the events of N metabolism or N translocation
are followed over short-term periods (days to weeks). The 15 N labeling
method involves a large enrichment of 15 N over background, making measurement
of isotopic effects easy because the difference between the isotopic compositions of
the source and the plant or the soil is large. In the 15 N natural abundance or d 15 N
method, plant tissues are analyzed to determine 15 N at naturally occurring levels
(d 15 N values, %). In the d 15 N method, there is a small enrichment of 15 N over
background, making measurement of isotopic effects difficult. The difference
between isotopic compositions of the source and the plant or soil is small, but
does reflect naturally occurring 15 N variations over long-term periods (years to
decades) (Hobbie and Hobbie 2006). However, variations of d 15 N values are often
small ( 10%) among AM or EM plant species that have unique physiological
processes in different habitats (Handley and Scrimgeour 1997; Högberg 1997;
Hobbie and Hobbie 2006). As a consequence, it is hard to demonstrate plantto-plant
N transfer through CMNs by d 15 N variations of mychorrhizal plants, at
least at face value, although they may demonstrate the magnitude of 15 N fractionations
during N translocation in long-term natural conditions.
17.3 Experimental Design for Investigating Two-Way
Nitrogen Transfer Between Plants
Here we explore the 15 N enrichment method to determine two-way N transfer
between plants through mychorrhizal connections and to measure the net benefit
to the partners. With either the N2-fixing or the non-N2-fixing plant as the N-donor,
four reciprocal pairings are created to study two-way plant-to-plant N transfer, and
to distinguish N transfer between the soil and the mychorrhizal pathway
(Table 17.1). First, as a control, the non-nodulated/non-mychorrhizal pair monitors
N transfer in the absence of both hyphal connections and N2 fixation. Second, the
nodulated/non-mychorrhizal pair tests effects of N2 fixation on N transfer in the
absence of hyphal connections. Third, the non-nodulated/mychorrhizal pair determines
effects of hyphal connections on N transfer in the absence of N 2-fixation.
Fourth, the nodulated/mychorrhizal pair examines effects of both mychorrhizal and
N2 fixation on N transfer. To minimize mass flow and diffusion through soil
pathways, two perforated Perspex plates (2.5 mm thick) are inserted in the middle
of a 5 l plastic box (300 12 150 cm) to divide the box into two chambers with a
5-mm air gap (Fig. 17.1). On one side of the two plates, sheets of 25–37 mm nylon
mesh are inserted on the inner side of the plates to allow only hyphal connections.
To further minimize water movement across the air gap between the two chambers,
high water holding capacity crystals [0.5% (w/w), RainSaver, Hortex Australia Pty.
Ltd., NSW, Australia] are incorporated in the potting mix. External 15 N may be
added directly to the N-donor side.