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Keywords:

  • nutrient exchange;
  • P efflux;
  • P transfer;
  • P translocation;
  • polyphosphate;
  • Gigaspora margarita;
  • arbuscular mycorrhiza

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  •  The enzymatic separation of physiologically active intraradical hyphae of arbuscular mycorrhiza (AM) fungi from host tissue is an effective way to analyse the physiological characteristics of intraradical hyphae.
  •  Applying this technique, first, both biomass and phosphorus (P) distribution were quantified in the intraradical and extraradical part of Gigaspora margarita colonizing onion. Second, the separated intraradical hyphae were incubated in vitro; efflux of phosphate from the hyphae and the hyphal polyphosphate (poly P) content were determined.
  •  A substantial proportion of P in mycorrhizal roots was fungal P, while the proportion of fungal biomass to root biomass was < 2%. Phosphate efflux and the decrease in poly-P content in the hyphae were both enhanced by the addition of glucose and 2-deoxyglucose, an analogue of glucose. The degrees of enhancement for phosphate efflux and poly-P decrease were comparable, suggesting that P efflux from intraradical hyphae was coupled with poly-P hydrolysis.
  •  Based on these findings, the translocation of P from fungus to host was estimated in relation to the distribution of hyphal P in extraradical and intraradical parts of the AM fungus.

Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

In arbuscular mycorrhiza (AM), nutrient exchange between fungi and host is one of the most important functions in the symbiosis. Recent advances in biochemical and molecular biological techniques have revealed a great amount of new information on this topic (Smith & Read, 1997; Harrison, 1999a,b; Jakobsen, 1999; Pfeffer et al., 1999; Saito, 2000). This new information has largely reinforced the ideas concerning symbiotic carbon (C)-phosphorus (P) transfer proposed by classical works in the 1970s and 1980s (Tinker, 1975; Harley & Smith, 1983). However, we are still far from a full understanding of the biochemical mechanisms of nutrient exchange in this symbiosis. The following view of phosphorus transfer from fungus to host plant has been widely accepted (Smith & Read, 1997). Phosphate in the soil solution is absorbed via a phosphate transporter in the extraradical hyphae (Harrison & van Buuren, 1995). The absorbed phosphate is condensed into polyphosphate (poly-P) and translocated by protoplasmic streaming into the intraradical hyphae (Cox et al., 1975; Cooper & Tinker, 1981). Finally, the poly-P may be hydrolysed and released as phosphate across the fungal membrane, probably at the arbuscule.

The separation of endosymbiont from host tissue can be a powerful tool, especially for those endosymbionts that cannot be independently cultured in vitro. For example, the isolation of mycetocytes, specialized host cells of aphids containing the endosymbiotic bacteria Buchnera, and the isolation of bacteroids from root nodules in leguminous plants, have been applied to molecular physiological studies of the endosymbiont (Ishikawa, 1989; Tajima & Kouchi, 1990). It is not easy to separate the intraradical hyphae of arbuscular mycorrhizas from host tissue because of the complex penetration of arbuscular hyphae into the cortex. However, Saito (1995) developed a method of isolating metabolically active intraradical hyphae from arbuscular mycorrhizal onion roots by enzymatic digestion with cellulase and pectinase. The hyphae isolated by this protocol were used for a radiorespirometric assay (Solaiman & Saito, 1997). Evolution of 14CO2 was linear with time for at least 6 h after the addition of 14C-substrates to the separated hyphae, suggesting that the respiratory activity of the isolated hyphae continued after isolation. This finding agrees with the in vivo NMR study by Shachar-Hill et al. (1995). Thus, this protocol for the separation of intraradical hyphae enables us to examine the physiological characteristics of intraradical hyphae involved in symbiotic nutrient exchange in vitro.

The first objective of the present study was to analyse the distribution of fungal biomass and fungal P pool sizes in intraradical and extraradical parts of the AM fungi by using the enzymatic separation technique. For this analysis we used part of the dataset published elsewhere by Solaiman et al. (1999). The second objective was to reproduce the C-P exchange in vitro using the separated intraradical hyphae. P efflux from intraradical hyphae was examined with or without the addition of glucose. To clarify the effect of glucose phosphorylation on P efflux, an analogue of glucose, 2-dexoxy glucose, was also examined, because this compound can be phosphorylated to glucose-6-phosphate but cannot be further metabolized in cells (Griffin, 1994). Poly-P contents in the hyphae during incubation were also examined. Based upon these experiments, we estimated P transfer and translocation from the AM fungi to the host.

Materials and Methods

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Experiment 1: distribution of fungal biomass and P in extraradical and intraradical parts of Gigaspora margarita

Host plant and mycorrhiza production

Seedlings of onion (Allium cepa L. CV. Sensyu-Chukou) plants were inoculated with Gigaspora margarita Becker & Hall MAFF 520054 and grown in a 2-l container under growth chamber conditions as described previously (Solaiman & Saito, 1997).

Plant biomass, mycorrhizal colonization and spore density

Six and 9 wk after transplanting, the shoots and roots in each container were collected. Roots were washed free of soil particles and organic debris. The f. wt of roots and shoots was measured. The root samples were divided into subsamples, 1 g of which was excised in order to assess the percentage of AM fungal colonization. The roots were stained with trypan blue in lactoglycerol (0.5 g l−1) according to the method of Phillips & Hayman (1970), and the percentage colonization of roots was estimated by the grid-line intersection method (Giovannetti & Mosse, 1980). Other root samples were used for separating the intraradical hyphae by means of enzymatic digestion, as described in the following section. Spores of G. margarita in the container were collected by the wet-sieving and decanting method described by Daniels & Skipper (1982), and the spores attached to the roots were also collected manually with fine forceps under a dissecting microscope. The number and the f. wt of the spores were measured.

Separation of intraradical hyphae and collection of extraradical hyphae

Roots were cut into 5 mm segments and digested with cellulase and pectinase, as described previously (Solaiman & Saito, 1997). Percoll discontinuous centrifugation was not performed in this study because the hyphae collected without this process were almost free of plant cells and the small amount of root tissue remaining was judged to be negligible for the present purpose. Extraradical hyphae were collected by the wet-sieving and decanting method. One hundred ml of fresh root-free substratum with three replicates from one container was sieved through a 106-µm sieve. The hyphae on the sieve were collected, and spores and debris were discarded by using fine forceps under a dissecting microscope. The f. wt of the hyphae was recorded by carefully removing excess moisture with small pieces of filter paper.

Measurement of hyphal biomass

Subsamples of the isolated intraradical hyphae and extraradical hyphae were fixed with 1 M formaldehyde solution and stained with trypan blue (0.5 g l−1, w/v). A part of the stained hyphal suspension was diluted with sterile distilled water and filtered on nitrocellulose membrane filters, which were then air-dried and immersed in microscopic oil for transparency. Hyphal length was measured by the grid-intersection method (Newman, 1966). For the intraradical hyphae, thick hyphae including intracellular hyphae and arbuscular trunks were measured, but the fine arbuscular branches were not evaluated. Extraradical hyphae of G. margarita were identified as strongly staining, angular, and aseptate (Bethlenfalvay & Ames, 1987). Intraradical hyphal length per root weight was also measured by using the subsamples of stained roots used for the colonization assay. This indicated the recovery efficiency of intraradical hyphae through the isolation process. All measurements were taken in triplicate.

Determination of total P in plants and fungi

For total P analysis of plants, the tissues (c. 2 g f. wt) were oven-dried and ground into fine particles. Fifty to 100 mg of ground tissue were digested with sulphuric acid (H2SO4) and hydrogen peroxide (H2O2) as an oxidant (Mizuno & Minami, 1980). Freshly collected intraradical or extraradical hyphae (c. 20–40 mg f. wt) or spores were also digested via the same method. Inorganic phosphate in these digests was analysed by the molybdenum blue method (Watanabe & Olsen, 1965).

Experiment 2: P efflux from the intraradical hyphae separated from roots

Assay of P efflux from intraradical hyphae in vitro

Mycorrhizal plants were grown as in experiment 1. After transplanting (6 wk), the intraradical hyphae were separated as described in the above section. The hyphae separated from 3 g fresh root per sample were suspended in 1 ml of washing buffer (10 mM Tris-HCl, pH 7.4, 0.3 M mannitol, 1 mM DTT) and incubated with or without 5 ml of 20 mM glucose or 2-deoxyglucose in 50 mM Tris-HCl buffer (pH 7.6) in a 50-ml beaker at 25°C for up to 4 h in a reciprocal shaker (120 strokes min−1). The buffer was collected hourly by means of centrifugation (3000 g, 5 min in 10 ml tube with slow acceleration and slow brake). After centrifugation, 5 ml of supernatant was collected by micropipette and replaced with new buffer. The supernatant was filtered with a 0.2-µm membrane filter immediately after collection. The inorganic phosphate concentration in the buffer was measured by taking 0.5 ml in a 2-ml microtube from each vial with EnzChek™ phosphate assay kit (Molecular Probes, Inc. OR, USA).

Polyphosphate contents in hyphae

The polyphosphate (poly-P) content in intraradical hyphae before and after incubation was determined colourimetrically with toluidine blue from successive extraction with trichloroacetic acid (TCA), EDTA, and phenol-chloroform (PC) according to the method of Solaiman et al. (1999).

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Experiment 1: distribution of fungal biomass and P in extraradical and intraradical parts of Gigaspora margarita

Percent root length colonized was 64% at 6 wk and 73% at 9 wk after transplanting. Distribution of fungal biomass together with plant biomass is shown schematically in Fig. 1 (f. wt basis). Total biomass of both the host plant and the mycorrhizal fungi increased about twofold during the 3 wk period. The intraradical hyphal biomass was < 2% of the root biomass. The ratio of the intraradical and the extraradical parts of G. margarita was about 1 : 1 at both sampling times, 1 : 0.83 at 6 wk and 1 : 0.89 at 9 wk after transplanting. Spore biomass increased four times while extraradical hyphae biomass increased only 1.5 times. The length of extraradical hyphae was 270 m per pot at 6 wk and 388 m per pot at 9 wk.

image

Figure 1. Distribution of biomass in compartments of Gigaspora margarita in symbiosis with onions at 6 and 9 wk after transplanting (mg f. wt per pot). Data are means of three with standard deviation.

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Total P contents in the intraradical and extraradical hyphae were 6.3 and 3.7 mg P g−1 f. wt respectively at 6 wk and 2.5 and 1.0 mg P g−1 f. wt at 9 wk. Spore P was 0.8 mg P g−1 f. wt. The distribution of P in the symbiotic system differed from the biomass distribution (Fig. 2). Total P content in the hyphae was high, so a large proportion of P in the AM system was retained in the hyphae. An especially high proportion of P in the roots was retained in the intraradical hyphae (80% of total P at 6 wk and 35% at 9 wk), in spite of the small proportion of biomass as hyphae. The ratio of P in the intraradical and extraradical parts of G. margarita was about 3 : 1 at both sampling times, 1 : 0.35 at 6 wk and 1 : 0.32 at 9 wk after transplanting. As sporulation increased, the proportion of P retained in the spores increased, while the proportion of P in the extraradical hyphae declined: at 9 wk it was 40% of that at 6 wk. The total amount of P retained in fungal cells did not change over the same period but the fungal biomass doubled in the same period.

image

Figure 2. Distribution of phosphorus in extraradical and intraradical parts of Gigaspora margarita and in onions 6 and 9 wk after transplanting (µg P per pot). Phosphorus in roots was estimated by subtracting fungal P from total P of mycorrhizal roots. Data are means of three with standard deviation.

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Experiment 2: phosphate efflux from isolated intraradical hyphae

In all treatments, phosphate efflux was almost linear with time at least within the 4 h incubation period (Fig. 3). Even with the control treatment, a large amount of phosphate was effluxed from the hyphae. However, the addition of glucose or 2-deoxyglucose significantly increased phosphate efflux. No significant difference between glucose and 2-deoxyglucose was found. The increase in phosphate efflux caused by the addition of these sugars was about 3.5 µg P g−1 fresh hyphae h−1.

image

Figure 3. Phosphate efflux from the intraradical hyphae of Gigaspora margarita in vitro. Cont, control treatment (diamonds); Glc, glucose (circles); 2dGlc, 2-deoxyglucose (triangles). Different letters in the figure indicate significant differences. P ≤ 5%.

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Polyphosphate contents in the intraradical hyphae before and after incubation (Fig. 4)

image

Figure 4. Polyphosphate contents in the intraradical hyphae of Gigaspora margarita before and after 4 h incubation, estimated by successive extraction. Cont, control treatment; Glc, glucose; 2dGlc, 2-deoxyglucoese; PC, Phenol-chloroform soluble fraction (Black columns); EDTA, EDTA soluble fraction (light grey columns); TCA, Trichloroacetic acid soluble fraction (dark grey columns). Different letters in the figure indicate significant differences by LSD. P ≤ 5%.

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The proportion of the three fractions (trichloroacetic acid (TCA) soluble, EDTA soluble and phenol-chloroform (PC) soluble), was about 40 : 35 : 25 and was not significantly different between the treatments. This ratio was within the range of that in our previous report (Solaiman et al., 1999). A decrease in poly-P content was found during incubation without the addition of sugars, but the decrease was significantly stimulated with the addition of either sugar. The decrease caused by the addition of the sugars was about 3.7 µg P g−1 fresh hyphae h−1, and no significant difference was found between glucose and 2-deoxyglucose.

Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Distribution of hyphal biomass and P in extraradical and intraradical parts of an AM fungus

This is the first report on the distribution of fungal P in the extraradical and the intraradical parts of AM fungi, although a large number of reports on P transfer and P translocation in AM have been published (Smith & Read, 1997). The hyphal biomass of AM fungi has been investigated by means of microscopic, chitin and phospholipid analysis (Tinker, 1975; Hepper, 1977; Bethlenfalvay et al., 1982; Toth et al., 1991; Olsson et al., 1999). Assuming that the moisture contents of fresh hyphae and roots were almost the same, the proportion of the hyphal mass in roots in the present study was lower than that reported by Hepper (1977) and that reported by Toth et al. (1991) but was within the range reported by Olsson et al. (1999). We obtained the specific root length of the onion, about 3 m g−1 f. wt basis, and specific hyphal length of 5.2 km g−1 hyphae from published data (Solaiman et al., 1999). Use of these conversion factors gave 23 m extraradical hyphae m−1 colonised root at 6 wk and 12 m m−1 at 9 wk. These data are within the range of 71 m m−1 for effective Glomus spp. and 7.1 m m−1 for ineffective Scutellospora calospora in onion (Sanders et al., 1977).

In the present study, the P contents in the hyphae were obtained on a fresh weight basis. To compare our data with data on a d. wt basis in the literature, we assumed that the hyphal moisture content was 80%. The estimated hyphal P concentration determined on a dry basis ranged from 1.9 to 3.2% for the intraradical part and from 0.5 to 1.3% for the extraradical part. These hyphal P concentrations were higher than those reported for other fungi, i.e., 0.5% to 2% P in ectomycorrhizal fungi in culture (Cairney & Smith, 1992; Ashford et al., 1994). To our knowledge, there is only one report on the P content in AM fungal hyphae. In this study by Capaccio & Callow (1982), it was suggested that the intraradical hyphae of G. mosseae is about 0.6% P; however, they did not provide any experimental values. In the present study, the total P concentrations in the intraradical hyphae were much higher than 0.6% and were almost equivalent to those in poly-P accumulating bacteria in activated sludge (Nakamura et al., 1991). Even if the P concentration in the intraradical hyphae was slightly overestimated because a certain amount of host contamination during the hyphal isolation process is inevitable, we can be certain that a large proportion of P in mycorrhizal roots was retained in the intraradical hyphae.

Another important finding is that the amount of P retained in the fungal cells did not change between 6 and 9 wk although the fungal biomass doubled during the same period. It has been shown previously that only a relatively small portion of AM fungal cells were active when examined with viable staining methods (Kough et al., 1987; Schubert et al., 1987; Sylvia, 1988; Hamel et al., 1990; Saito et al., 1993). Arbuscules tend to become senescent quickly and degenerate within 1–2 wk (Cox & Tinker, 1976; Toth & Miller, 1984). Assuming that only active fungal cells retain high P concentrations, the present results agree with these observations and imply that only a small fraction of the intraradical hyphae are involved in P translocation.

Phosphate efflux from the intraradical hyphae separated from roots

This study has demonstrated phosphate efflux from intraradical hyphae in vitro for the first time. A large amount of phosphate was effluxed during 4 h of incubation even without the addition of glucose. The intraradical hyphae collected by the method used in the present study were physiologically active but were already fragmented, so some cytoplasmic phosphorus may have leaked out and been hydrolysed by phosphatases (Joner & Johansen, 2000) during the incubation. However, it is possible that the separated hyphae had the potential to efflux phosphate themselves without any C sources. P efflux was partly inhibited by cyanide, which is an inhibitor of respiratory activities (unpublished results), suggesting that part of the P efflux was not leakage from damaged tissue.

It is significant that both glucose and its analogue, 2-deoxyglucose, enhanced phosphate efflux. At the same time, hyphal poly-P content decreased with the addition of the sugars. Furthermore, the enhancement of phosphate efflux caused by the sugars, 3.5 µg P g−1 fresh hyphae h−1, was comparable to the decrease of hyphal poly-P content, 3.7 µg P g−1 fresh hyphae h−1, suggesting that the addition of sugars increased poly-P hydrolysis and that the phosphate released was effluxed. An analogue of glucose, 2-deoxyglucose, can be absorbed and phosphorylated to glucose-6-phosphate by fungal cells but cannot be further metabolised. Therefore, at least a part of the phosphate efflux from the intraradical hyphae was closely related to glucose uptake and its subsequent phosphorylation (Woolhouse, 1975). In fact, phosphotransferase systems for sugar transport are common among fungi, but there is a diversity of transport systems (Griffin, 1994).

The role of alkaline phosphatase (ALPase) expressed in arbuscules in relation to the C-P exchange has been disputed (Gianinazzi et al., 1979; Tisserant et al., 1992; Larsen et al., 1996). Larsen et al. (1996) found that benomyl inhibited P translocation through hyphae but did not inhibit ALPase; however, the inhibition of ALPase with benomyl has been reported elsewhere (Thingstrup & Rosendahl, 1994). However, inhibition of overall P translocation by means of benomyl may not necessarily indicate that ALPase is not involved in phosphate efflux from the intraradical hyphae. Ezawa et al. (1999) investigated the characteristics of ALPase extracted from intraradical hyphae of G. etunicatum. They hypothesised that some of the phosphate released from intraradical hyphae may be provided by ALPase via hydrolysis of intermediary sugar phosphates because of its low Km to sugar phosphate compounds and its inability to hydrolyse pyrophosphate compounds. Assuming that ALPase is indeed involved in phosphate efflux, the present findings suggest that a part of glucose-6-phosphate derived from the absorbed glucose is hydrolysed with the ALPase and that the phosphate is then effluxed. For further studies, stoichiometric approaches to absorbed C and effluxed P and specific inhibitors for transport processes and other analogue compounds of glucose should be used.

Based upon the localisation of H+-ATPase, it has been suggested that phosphate efflux may occur in arbuscular hyphae while carbon compounds may be absorbed in the intercellular hyphae (Gianinazzi-Pearson et al., 1991; Smith & Read, 1997). The present results show that these processes may be closely related to each other and may be recognised as a coupled exchange process. However, we have no experimental observations so far that indicate whether phosphate efflux and C absorption occur at the same site.

Estimation of P transfer based upon Experiments 1 and 2

We have now estimated the hyphal P distribution in the AM system and the phosphate efflux from intraradical hyphae in vitro. Based upon these data and the following assumptions, we estimated the translocation and transfer of P from soil to host plant during the period from 6 to 9 wk after transplanting.

  • 1)
     P in the fungus was composed of three pools: total P excluding poly-P, poly-P and spore P.
  • 2)
     Translocation of P from the extraradical to the intraradical part and transfer of P from the fungus to host passed through the poly-P pool in each part. Although poly-P was composed of various chain lengths, poly-P pools were homogenous.
  • 3)
     The P transfer from the intraradical hyphae to the host was equivalent to the increase in phosphate efflux caused by the addition of glucose in experiment 2. The phosphate efflux was constant from 6 to 9 wk.

Poly-P contents in the hyphae were presented in our previous report (Solaiman et al., 1999). The amount of P given to the host by the fungus was estimated by the following formula. The original formula was developed for estimation of nutrient inflow through growing roots (Nye & Tinker, 1977). Instead of length, we used hyphal fresh weight since the hyphal specific length was almost constant during the growing period (Solaiman et al., 1999).

  • image(Eqn 1 )

(W1, W2, fresh weight of intraradical hyphae at harvesting time T1 and T2, respectively; ΔP, P transfer from fungus to host during the 3 wk (P mg pot−1).) Incorporating W1, 99 mg pot−1, W2, 231 mg pot−1, T2−T1, 3 wk, and P efflux, 3.5 µg P g−1 fresh hyphae h−1 from experiment 2, ΔP, 275 mg P pot−1 was obtained.

Figure 5 schematically represents the estimated P translocation and transfer in AM symbiosis. Vigorous protoplasmic streaming has been observed in the extraradical hyphae of AM fungi, and its movement is bi-directional (Cine-document, 1998). Therefore, the flow of P translocation in Figure 5 indicates the net flow. This figure demonstrates several interesting points. First, about 12% of P absorbed by the host plant was received from the fungus. This is markedly lower than the values obtained by the hyphal compartment experiments (Li et al., 1991a,b; Pearson & Jakobsen, 1993). However, in those compartment experiments, available P in the root compartment tended to be depleted so that the contribution of hyphae to total P uptake may have been higher than that under non-compartment growing conditions as in the present study.

image

Figure 5. Estimated phosphorus flow in arbuscular mycorrhizal onion roots between 6 and 9 wk after transplanting (µg P per pot). Numbers in italics are the estimated values. Numbers in roman are values obtained in the experiments or from the literature. The experimental values are at 6 (top) and 9 (bottom) wk, respectively.

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Second, the turnover of the poly-P pool was estimated as follows.

  • image(Eqn 2)

By using the mean value of the poly-P pool size at 6 and 9 wk, the turnover was estimated to be about 0.74 day−1 for extraradical hyphae and 0.32 day−1 for intraradical hyphae. It is still uncertain if poly-P is the only form of P translocation. Glomus manihotis was found to contain no poly-P granules in its hyphae based upon a DAPI stain (Boddington & Dodd, 1999). However, the hyphae of G. margarita contained substantial amounts of poly-P (Solaiman et al., 1999), and the poly-P contents in the hyphae of G. coronatum were found to depend on P availability in the culture medium (Ezawa et al., 2001). Therefore, the fast turnover of poly-P is not surprising.

Third, we estimated P inflow in the extraradical hyphae according to the following formula (Nye & Tinker, 1977):

  • image(Eqn 3)

(L1, L2, length of extraradical hyphae at harvesting time T1 and T2, respectively; ΔP, estimated phosphorus absorption by the extraradical hyphae (187 µg pot−1 3 wk−1).) Incorporating L1, 270 m pot−1, L2, 388 m pot−1, T2−T1, 3 wk, a P inflow of 10.9 f mol P m−1 s−1 was obtained. This value was within the range of previous estimates (1–430 f mol P m−1 s−1) (Tinker, 1975; Li et al., 199a; Jakobsen et al., 1992) and was most similar to that estimated for indigenous AM fungi of field-grown wheat (38 f mol P m−1 s−1, Schweiger & Jakobsen, 1999). We used the fragmented hyphae separated from roots, so the actual P transfer in situ might be higher than the experimental values obtained in vitro. This suggests that the actual P transfer may not be very different from the values obtained in the present in vitro experiment. In the present study we did not evaluate arbuscular surface area, so we could not estimate P flux across the arbuscular surface (Smith et al., 1994).

The present study is the first report on the distribution of fungal P in extraradical and intraradical parts of the G. margarita colonising onion, and on the phosphate efflux from the intraradical hyphae separated from host roots and its enhancement with glucose. Based upon these data and a number of assumptions, we estimated the translocation and transfer of P in AM symbiosis. Our estimates more or less agree with the estimates previously arrived at by other methodologies. In conclusion, by applying the technique of separating intraradical hyphae from host roots, we were able to clarify the translocation and transfer of P in AM symbiosis in a quantitative manner. Further investigation of phosphate efflux using the present experimental system will shed light on the mechanisms of nutrient exchange.

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

The study was supported in part by Promotion of Basic Research Activities for Innovative Biosciences (PROBRAIN), Bio-oriented Technology Research Advancement Institution. The authors are grateful to T. Kojima for her assistance in raising the mycorrhizal plants and to Prof. R. Koide for his critical reading of this manuscript. MZS is grateful to Japan Science and Technology Corporation for providing his fellowship.

References

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
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