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- Materials and Methods
The role of mycorrhizas in nutrient uptake from soil is well established, but their role in transferring nutrients among plants is less well understood. While many studies demonstrate resource movement between the same or different plant species that share the same mycorrhizal type (arbuscular mycorrhizal, AM or ectomycorrhizal, EM), resource exchange between plants with different mycorrhizal types is not well established. Transfer of carbon, nutrients, and water among plants that are EM or AM has been demonstrated (Brownlee et al., 1983; Finlay & Read, 1986a, 1986b; Newman, 1988; Newman et al., 1992; Simard et al., 2002; He et al., 2003; Leake et al., 2004). Transfers among or between plants of the same mycorrhizal type may occur through mycorrhizal networks that redistribute resources among plants along source–sink gradients (Francis & Read, 1984; Newman, 1988; Bethlenfalvay et al., 1991; Newman et al., 1992; Frey & Schuepp, 1993; Simard et al., 1997; Smith & Read, 1997; Perry, 1998; Lerat et al., 2002; Carey et al., 2004; Leake et al., 2004).
Soil nutrient uptake, particularly N, is usually considered to be unidirectional from soil to roots to shoots (Marschner, 1995; Brady & Weil, 2002). In contrast, C, nutrients and water transfers can be bidirectional between different plant species through mycorrhizal networks, as shown in field, growth-chamber and glasshouse studies using isotopes (Duddridge et al., 1980; Chiariello et al., 1982; Francis & Read, 1984; Newman, 1988; Eissenstat, 1990; Hamel et al., 1992; Newman et al., 1992; Ek et al., 1996; Johansen & Jensen, 1996; Lerat et al., 2002; Simard et al., 2002; He et al., 2003, 2004, 2005; Querejeta et al., 2003; Leake et al., 2004; Simard & Durall, 2004).
Although such transfers are well documented, the quantities transferred are generally low and the ecological implications of sharing or competing for these resources among AM and EM plants are less well known (Newman, 1988; Perry et al., 1998; Robinson & Fitter, 1999; Simard et al., 2002; He et al., 2003; Leake et al., 2004; Simard & Durall, 2004). Pulse-chase experiments lasting a few days may not reflect transfers that would occur over longer time periods. Complex AM and EM networks in blue oak woodlands encourage competition for soil resources (Welker et al., 1991; Cheng & Bledsoe, 2004) and water (Gordon & Rice, 1993). Plant species with AM and EM mycorrhizal types may interact in complicated ways to exchange nutrients and water. Plants sharing the same mycorrhizal species have the potential to share soil resources directly via fungal hyphae. Sharing of nutrients has been reported for C (Francis & Read, 1984; Newman, 1988; Simard et al., 1997; Leake et al., 2004); N (Newman, 1988; Simard et al., 2002; He et al., 2003, 2004, 2005; Simard & Durall, 2004); P (Chiariello et al., 1982; Johansen & Jensen, 1996); and water (Brownlee et al., 1983; Querejeta et al., 2003).
In California oak woodlands, plants are highly dependent on mycorrhizae for growth and survival. A major species in these woodlands is blue oak (Quercus douglasii Hook. & Arn.) that forms predominantly EM associations, but AM symbionts have also been observed (Egerton-Warburton & Allen, 2001; Douhan et al., 2005). Other major species in these woodlands include gray pine (Pinus sabiniana Douglas) that forms EM associations, and buckbrush [Ceanothus cuneatus (Hook.) Nutt., Rhamnaceae] and annual species that form AMs (Rose & Youngberg, 1980; Cheng & Bledsoe, 2002a; Douhan et al., 2005). In this oak woodland community with its mixed mycorrhizal associations, we measured 15N movement from gray pine saplings to nearby saplings of gray pine and blue oak, and to buckbrush shrubs as well as to adjacent annuals: hedgehog dogtail (Cynosurus echinatus L.), hedge parsley (Torilis arvensis (Hudson) Link), and rose clover (Trifolium hirtum All.). Our goal was to determine whether mycorrhizal type (EM or AM) affects N transfer between plant species growing in a Mediterranean oak savanna ecosystem. We designed a field study to examine three questions: (1) Does N move from an EM host to nearby EM plants? (2) Does N move from an EM host to nearby AM plants? and (3) Does N transfer occur preferentially from EM donors to EM receivers?
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- Materials and Methods
The goal of pulse labeling is to follow the path of 15N without significantly altering the N status of the target species. In our study the total N (Table 1) and NDSF (Table 2) data indicate that, in this experiment, this goal was accomplished. Except for the decrease in total N accompanying senescence of annuals, total N was constant throughout the 4-wk experiment in both roots and foliage of all woody species. Foliar N levels in the species sampled were low but not deficient. In Mediterranean climates mycorrhizal associations are known to be important for nutrient and water relations of native species; the plants in our experiment were symbiotic with both EM and AM fungi (Table 1). This difference in mycorrhizal status allowed us to compare N movement between and among plants with these two mycorrhizal types. We found that annuals, oak saplings and buckbrush formed AM fungal associations, and that the oaks and pines formed EM associations. These findings confirm literature reports that oaks are predominantly EM but can form AM, especially when young (Rothwell et al., 1983; Egerton-Warburton & Allen, 2001; Valentine et al., 2002). Cheng & Bledsoe (2002a, 2002b) reported that EM colonization of blue oak roots at a nearby site fluctuated seasonally from a low of 0–10% in the dry season to a high of 80% in the wet season. We conducted our study in the spring (cool and wet) when roots and mycorrhizae are more active. In a preliminary study we determined that 15N natural abundance values in pines, forbs and grasses changed little over a 4-wk period in spring (data not shown). In this study we did not identify the fungal species on roots. However, in other studies at our field site the most abundant EM genera associated with blue oak were Cenococcum, Tuber, Tomentella and Sebacina (Cheng & Bledsoe, 2002a; Douhan et al., 2004; M. Smith, personal communication). The most abundant AM genus was Glomus (Douhan et al., 2005).
15N movement from EM donors to EM and AM receivers
Both EM and AM plants in close proximity to labeled gray pine acquired 15N during the 4-wk experimental period. Annual plants growing near the labeled pines became enriched rapidly and contained significant 15N concentrations 2 wk following 15N application. Roots and needles of gray pine saplings within 1 m of the donor pines also became enriched. The increased 15N content of receiver pine roots and shoots demonstrates that pine transferred N to other pines. However, this N was not transferred preferentially to other pines. 15N at % excess was greater in receiver oak saplings than in receiver pine and buckbrush sapling. The NDFS was lower in oaks, showing that the amount of N transferred comprised a smaller quantity of the blue oak total N pool compared with gray pine and buckbrush. In contrast, δ15N values were greater in gray pine and blue oak receivers than in buckbrush saplings because of different background δ15N values. Annuals had greater 15NDFS, 15N at % excess and δ15N values than perennial receivers, showing that the total N pool of the annuals was more affected by translocation of N from the donor pine. The larger NDFS in annuals may be related to a smaller total N pool in the annuals, such that a small amount of 15N could significantly alter the NDFS.
Nitrogen transfer between plants can take place by direct or indirect routes (McNeill & Wood, 1990). Direct N transfer occurs through mycorrhizal connections among plants (Newman, 1988; Frey & Schuepp, 1993; Simard et al., 2002; He et al., 2003, 2004, 2005; Leake et al., 2004; Simard & Durall, 2004). However, amounts of N transferred were low and may not be ecologically relevant (Newman, 1988; Simard et al., 2002; He et al., 2003; Leake et al., 2004; Simard & Durall, 2004). Direct links between EM and AM are unlikely: AM and EM fungi are taxonomically distinct and cannot fuse their hyphae and exchange/transfer materials.
Nitrogen transfer from EM pines to AM receivers shows that direct fungal connections are not necessary for N transfer among plants in this oak woodland community. Other indirect mechanisms must be operative. Nitrogen transfer from donor roots could be explained by at least six processes: (1) turnover of mycorrhizal hyphae (Staddon et al., 2003); (2) N movement from mycorrhizal mycelia to soil (Johnson et al., 2002); (3) N foraging in the rhizosphere by receiver hyphae (Read & Perez-Moreno, 2003; Cairney, 2005); (4) root exudation (Jones et al., 2004); (5) faunal grazing (Klironomos & Hart, 2001; Perez-Moreno & Read, 2001; Johnson et al., 2005); and (6) recapture of N-containing materials from rhizodeposition (Stern, 1993; Dubach & Russelle, 1994; Chalk, 1996; Paynel et al., 2001; Walker et al., 2003) which, in turn, is regulated by interactions between plants and mycorrhizas (Jones et al., 2004). Our data document a rapid transfer among AM and EM plants, but do not allow us to determine which processes are responsible for the 15N transfers we observed.
The transfer of foliar 15N from the donor to its own roots and to roots of receivers and annuals occurred rapidly (Fig. 2). The steep increase in δ15N and the linear response between the second and the fourth week suggests that N transfer from donor roots continued at similar rates during the 4-wk sampling period. This ongoing N transfer may be a significant pathway for N exchange among plants. The roots and leaves of the annual plants had greater 15N derived from source (NDFS) and were more enriched (15N at % excess and δ15N values) than perennial receivers, irrespective of the mycorrhizal type of the receivers. Apparently annuals, with their extensive fibrous root systems, especially in upper soil layers, were a strong sink for N (Jackson et al., 1988; Cheng & Bledsoe, 2002a). Root systems of perennial species (blue oak, gray pine and buckbrush) are unevenly distributed and are deeper with less branching than annuals (Millikin & Bledsoe, 1999). The difference in root distribution may affect competition for soil resources such as water and N (Tilman, 1989; Welker et al., 1991; Gordon & Rice, 1993; Momen et al., 1994; Koukoura & Menke, 1995; Cheng & Bledsoe, 2004).
We measured 15N transfer against a concentration gradient – from pines with lower percentage N to grasses and forbs with higher percentage N. However, we did not measure two-way or net transfer, only one-way transfer. Recently, two-way or bidirectional N transfer has been documented from barley to pea (Johansen & Jensen, 1996) and from Eucalyptus to Casuarina (He et al., 2004, 2005). If two-way N transfer is a universal phenomenon, N transfer against a concentration gradient, for example from pine to oak, may occur.
Foliar labeling and N movement
Under controlled conditions, leaf 15N feeding is an efficient technique for labeling above- and below-ground plant biomass (Schmidt & Scrimgeour, 2001; Yasmin et al., 2006). Although labeling foliage using 15N to observe N movement below ground has not been reported extensively for field conditions (Horwath et al., 1992; Schmidt & Scrimgeour, 2001), this technique worked well at our oak woodland site. Movement of N in trees and other perennials has been thought of as a primarily unidirectional flow from roots to above-ground components during leaf expansion, to structural components during senescence, and, in early spring to fine roots from large roots as roots elongate (Krammer & Kozlowski, 1979). However, our results show that N movement can be a two-way dynamic process, and can occur from needles to roots in donors and from roots to needles in receivers, even after leaf expansion and early root growth. Our study examined the net transfer of 15N among AM and EM plants. We recognize that N transfer/turnover may have occurred several times during the 4-wk period. If that is the case, then we believe that a small pool of actively cycling N is responsible for N transfers among plants.
In summary, our results suggest that mycorrhizas play an important role in transferring N between plants, including release and recapture of N from the rhizosphere. Nitrogen transfer among plants in California oak woodlands expands our view of below-ground complexity associated with N cycling. Nitrogen translocation among perennial and annual plant species is more complex than expected. Nitrogen transfers occurred beyond the boundaries of a common mycorrhizal linkage among plants of the same species. Therefore indirect pathways, in addition to direct transfers, must be invoked to describe the complex N movement observed in the oak woodland ecosystem.