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Journal ArticleDOI

A 31P nuclear magnetic resonance study of phosphate levels in roots of ectomycorrhizal and nonmycorrhizal plants of Castanea sativa Mill.

TL;DR: The level of orthophosphate in mycorrhizal roots was significantly lower than in nonmycorrhIZal ones, which indicates that this system relies upon the fungal polyphosphates as a major source of phosphate.
Abstract: 31P-Nuclear Magnetic Resonance (NMR) was used to assess phosphate distribution in ectomycorrhizal and nonmycorrhizal roots of Castanea sativa Mill. as well as in the mycorrhizal fungus Pisolithus tinctorius in order to gain insight into phosphate trafficking in these systems. The fungus P. tinctorius accumulated high levels of polyphosphates during the rapid phase of growth. Mycorrhizal and nonmycorrhizal roots accumulate orthophosphate. Only mycorrhizal roots presented polyphosphates. The content in polyphosphates increased along the 3 months of mycorrhiza formation. In mycorrhizal roots of plants cultured under axenic conditions, the orthophosphate pool decreased along the culture time. In nonmycorrhizal roots the decrease in the orthophosphate content was less pronounced. The level of orthophosphate in mycorrhizal roots was significantly lower than in nonmycorrhizal ones, which indicates that this system relies upon the fungal polyphosphates as a major source of phosphate.

Summary (2 min read)

Introduction

  • The increase in phosphate concentration in mycorrhizal plants was also correlated with increased photosynthetic rates, as the phosphate level of the chloroplast influences photosynthetic rates (Osmond 1981; Coombs 1985; Foyer and Spencer 1986; Jakobsen 1991) .
  • It was suggested that phosphate is translocated along hyphae as polyphosphate granules inside vacuoles (Cox et al. 1980 ).

Materials and methods

  • Castanea sativa micropropagated plants were obtained as previously described (Martins et al. 1996) .
  • The fungus Pisolithus tinctorius (Pers.) Coker and Couch isolate 289/Marx kindly supplied by Dr. Ingrid Kottke (Tübingen University) was maintained on a MMN solid medium (Marx 1969) .

Mycorrhizal synthesis

  • Fungus was inoculated 3 weeks before plant transfer.
  • In vitro mycorrhization of micropropagated plants was induced 4-5 weeks after plant rooting, by transfer of rooted plants to the petri dishes inoculated 3 weeks before with mycelium.
  • A portion of the petri dishes with roots was covered with aluminum foil to prevent oxidation of root phenols by light.
  • Control plants were transferred to petri dishes without fungus and maintained under the same conditions.

Sample preparation for NMR

  • Samples of excised root systems of mycorrhizal or control plants as well as samples of pure cultures of the fungus were prepared for NMR analysis.
  • Similar masses of material and similar packing were used in the different experiments.
  • Two glass capillaries were fitted into the NMR tube, one positioned a few millimeters from the bottom (inlet of medium) and the other just above the cotton wool filter that was used to restrict the volume of the biological material (outlet of medium).
  • The acquisition time of each spectrum was approximately 40 min.
  • In all the experiments, the probe head temperature was kept at 25°C.

Phosphate quantification

  • Extraction of total P from roots leaves and stems of mycorrhizal and nonmycorrhizal plants was performed according to Bowman (1988) .
  • Phosphate quantification was made 30, 40, 60 and 90 days after mycorrhizal induction in vitro by the molybdenum blue method according to John (1970) .

Results and discussion

  • 31 P-NMR spectra of the fungus samples, either in pure culture or extramatrical hyphae collected from petri dishes containing 1-month-old mycorrhizas, showed the presence of resonances due to the vacuolar orthophosphate pool and the polyphosphates pool (Fig. 1 ) as al-ready reported for P. tinctorius (Ashford et al. 1994 ) and for other fungi (Martin et al.
  • When present in a P-limiting medium, the stored polyphosphates are hydrolyzed, releasing Pi and maintaining Pi concentration within the fungal cell (Martin et al. 1985; MacFall et al. 1992) .
  • Chestnut mycorrhizas are able to accumulate phosphate, mostly in the form of polyphosphates, in contrast to nonmycorrhizal roots, which accumulate only orthophosphate (Fig. 2 ).
  • The differences in phosphate content between mycorrhizal and nonmycorrhizal plants decreased with time.

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Citations
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Journal ArticleDOI
TL;DR: While large gaps remain in understanding of the physiological and molecular mechanisms that underpin movement of P in ECM mycelia in soil and P transfer to the plant, host P demand seems likely to be a key driver of these processes.
Abstract: Many forest trees have evolved mutualistic symbioses with ectomycorrhizal (ECM) fungi that contribute to their phosphorus (P) nutrition. Forest productivity is frequently limited by P, a phenomenon that is likely to become more widespread under future conditions of elevated atmospheric CO2 concentration [CO2]. It is thus timely that this review considers current understanding of the key processes (absorption, translocation and transfer to the plant host) in ECM fungus-mediated P nutrition of forest trees. Solubilisation of inorganic P (Pi) and hydrolysis of organic P by ECM fungi in soil occurs largely at the growing mycelial front, where Pi absorption is facilitated by high affinity transporters. While large gaps remain in our understanding of the physiological and molecular mechanisms that underpin movement of P in ECM mycelia in soil and P transfer to the plant, host P demand seems likely to be a key driver of these processes. ECM fungi may make considerable contributions to meeting the likely increased P demand of trees under elevated [CO2] via increased colonization levels, shifts in ECM fungal community structure and changed patterns of EMM production. Further research into the spatial scale of ECM-mediated P movements in soil, along with the interplay between ECM fungi and other soil microflora is advocated.

129 citations


Cites background from "A 31P nuclear magnetic resonance st..."

  • ...…with factors that include P availability and the relative growth rates of the host and ECM fungal partner, but as much as 90% of absorbed P can be retained in the fungal tissues of excised ECM root tips, mainly in the form of polyphosphates (Harley and McCready 1952, 1981; Martins et al. 1999)....

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Journal ArticleDOI
TL;DR: NMR has been useful in analysing metabolism, transport and energetics, and the results of such studies have practical and ecological significance.
Abstract: Summary Nuclear magnetic resonance (NMR) studies of mycorrhizal symbioses have illuminated a number of functional aspects of these complex associations. Here we review studies of the two main types of mycorrhiza (ectomycorrhizas and arbuscular mycorrhizas) to which NMR has been applied. Although the physiological questions addressed in each case are frequently the same, these two mutualistic symbioses are sufficiently different to justify separate discussion. In conjunction with isotopic labelling NMR is able to examine the transfer of substrates between the symbionts both in vivo and in vitro, as well as the production of secondary metabolites in response to colonization. In addition, this methodology is capable of determining the locations of the biosynthesis and translocations of storage compounds, such as polyphosphates, lipids and carbohydrates, in mycorrhizal fungi both in the free-living and in the symbiotic stages of their life cycle. NMR has been useful in analysing metabolism, transport and energetics, and the results of such studies have practical and ecological significance. Models of transport and physiology to which NMR has contributed form the necessary foundation for functional genomic exploration.

63 citations

Journal ArticleDOI
TL;DR: The hypothesis that polyP is the major P species translocated in the tubular vacuolar network, the presence of which was previously demonstrated in AM fungi, is supported.
Abstract: Summary • Polyphosphate (polyP) is presumably central to phosphate (P) metabolism of arbuscular mycorrhizal (AM) fungi, but its synthesis, location and chain lengths are poorly characterized. Here, we applied noninvasive and nondestructive nuclear magnetic resonance (NMR) spectroscopy to obtain novel information on AM fungal polyP. • In vivo31P NMR spectroscopy was used to characterize polyP and other P pools in external hyphae and in mycorrhizal roots of associations between Glomus intraradices and cucumber (Cucumis sativus). • A time-course study of P-starved external hyphae supplied with additional P showed that polyP appeared more rapidly than vacuolar inorganic P. These P metabolites also appeared in the roots, but later. PolyP considerably exceeded amounts of vacuolar inorganic P, where it was located in acidic, presumably vacuolar compartments, and had a short average chain length. • The rapid synthesis of polyP might be important for the maintenance of effective hyphal P uptake. Our data support the hypothesis that polyP is the major P species translocated in the tubular vacuolar network, the presence of which was previously demonstrated in AM fungi.

62 citations

Journal ArticleDOI
TL;DR: The 31P NMR spectra of excised AM fungi and mycorrhizal roots contained signals from polyphosphate (PolyP), which were absent in theSpectra of nonmycorrhIZal roots, demonstrating that the Pi taken up by the fungus was transformed into PolyP with a short chain length.
Abstract: 31P nuclear magnetic resonance (NMR) spectroscopy was used to study phosphate (P) metabolism in mycorrhizal and nonmycorrhizal roots of cucumber (Cucumis sativus L) and in external mycelium of the arbuscular mycorrhizal (AM) fungus Glomus intraradices Schenck & Smith. The in vivo NMR method allows biological systems to be studied non-invasively and non-destructively. 31P NMR experiments provide information about cytoplasmic and vacuolar pH, based on the pH-dependent chemical shifts of the signals arising from the inorganic P (Pi) located in the two compartments. Similarly, the resonances arising from α, β and γ phosphates of nucleoside triphosphates (NTP) and nucleoside diphosphates (NDP) supply knowledge about the metabolic activity and the energetic status of the tissue. In addition, the kinetic behaviour of P uptake and storage can be determined with this method. The 31P NMR spectra of excised AM fungi and mycorrhizal roots contained signals from polyphosphate (PolyP), which were absent in the spectra of nonmycorrhizal roots. This demonstrated that the Pi taken up by the fungus was transformed into PolyP with a short chain length. The spectra of excised AM fungi revealed only a small signal from the cytoplasmic Pi, suggesting a low cytoplasmic volume in this AM fungus.

56 citations


Cites background from "A 31P nuclear magnetic resonance st..."

  • ...…role in the transportation and storage of phosphorus in the fungus, and when P was added to ectomycorrhizal fungi, PolyP signals were found in the31P NMR spectra (Ashford et al., 1994; Gerlitz and Gerlitz, 1997; Gerlitz and Werk, 1994; Martin et al., 1983, 1985, 1994; Martins et al., 1999)....

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Journal ArticleDOI
TL;DR: The results support the existence of specific host plant effects on fungal P metabolism able to provide P in the apoplast of ectomycorrhizal roots, and specifically increased the proportion of short-chain polyP in the interacting mycelia.
Abstract: Ectomycorrhizal (ECM) association can improve plant phosphorus (P) nutrition. Polyphosphates (polyP), synthesized in distant fungal cells after P uptake may contribute to P supply from the fungus to the host plant if they are hydrolyzed to phosphate in ECM roots then transferred to the host plant when required. In this study, we addressed this hypothesis for the ECM fungus Hebeloma cylindrosporum grown in vitro and incubated without plant or with host (Pinus pinaster) and non-host (Zea mays) plants, using an experimental system simulating the symbiotic interface. We used 32P labelling to quantify P accumulation and P efflux and in vivo and in vitro NMR spectroscopy and cytological staining to follow the fate of fungal polyP. Phosphate supply triggered a massive P accumulation as newly synthesized long-chain polyP in H. cylindrosporum if previously grown under P-deficient conditions. P efflux from H. cylindrosporum towards the roots was stimulated by both host and non-host plants. However, the host plant enhanced 32P release compared to the non-host plant and specifically increased the proportion of short-chain polyP in the interacting mycelia. These results support the existence of specific host-plant effects on fungal P metabolism able to provide P in the apoplast of ectomycorrhizal roots.

21 citations


Cites methods from "A 31P nuclear magnetic resonance st..."

  • ...Signal identification was carried out according to chemical shifts reported in other ectomycorrhizal fungi (Gerlitz & Werk 1994; Martins et al. 1999)....

    [...]

References
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Journal ArticleDOI
TL;DR: A threshold effect (no increase in growth with increasing additions of P) characteristic of non-mycorrhizal seedlings was eliminated by mycorrhIZal infection, and the effect of myCorrhizas on seedling P status diminished with increasing soil P.
Abstract: Growth and phosphorus acquisition of pot-grown seedlings of karri (Eucalyptus diversicolor F. Muell.) were examined following inoculation with four ectomycorrhizal fungi -Descolea maculata Bougher (two isolates), Pisolithus tinctorius (Pers.) Coker & Couch, and Laccaria laccata (Scop, ex Fr.) Berk. & Br. Seedlings were raised in steam-sterilized sand to which 13 rates of phosphorus (0.100 mg P kg-1 soil) were applied. All fungi except P. tinctorius produced a plant growth response. L. laccata produced the largest growth response. Responses were greatest at low rates of application of P to soil. There was no effect of the fungi on growth at levels of P application above 28 mg P kg-1 soil. A threshold effect (no increase in growth with increasing additions of P) characteristic of non-mycorrhizal seedlings was eliminated by mycorrhizal infection. Mycorrhizal inoculation increased P content of plant tissues at sub-optimal levels of P supply. The effect of mycorrhizas on seedling P status diminished with increasing soil P. One isolate of D. maculata often had greater rates of P accumulation and produced higher concentrations of P in plant tissues than L. laccata, but did not produce greater plant biomass. Frequency of infection for all fungi was low in soils with no additional P, and greatest with the addition of 2 mg P kg-1 soil (L. laccata and D. maculata isolate A), or 4 mg P kg-1 soil (D. maculata isolate B). Infection was reduced with increasing soil P, and not evident at 36 mg P kg-1 soil or higher levels of soil P. L. laccata had higher infection frequency and mycorrhizal root length at all levels of soil P than the D. maculata isolates. Two fungi produced basidiomes. This occurred at levels of soil P application ranging from 4 to 28 mg P kg-1 soil for D. maculata (isolate B), and at 4 to 28 mg P kg-1 soil for L. laccata.

161 citations

Journal ArticleDOI
TL;DR: X-ray microanalysis has confirmed, and measured, the phosphorus content of cytochemically-demonstrated polyphosphate granules in onion mycorrhizas, and fulfils the hypothesis that phosphorus is translocated in the fungus by means of the transport of polyph phosphate granules by cytoplasmic streaming.
Abstract: Summary X-ray microanalysis has confirmed, and measured, the phosphorus content (0·03 g cm-3) of cytochemically-demonstrated polyphosphate granules in onion mycorrhizas. The volume occupied by the granules measured from stereoscopic high-voltage electron micrographs of thick sections was 0·8 % of the fungal volume, and cytoplasmic streaming rates of the extra-matrical mycelium in soil measured in the light microscope were around 12·6 cm h-1. From these data a flux rate of about 2·7 × 10-8 mol P cm-2 s-1 has been calculated, which fulfils the hypothesis that phosphorus is translocated in the fungus by means of the transport of polyphosphate granules by cytoplasmic streaming.

145 citations

Journal ArticleDOI
TL;DR: It is confirmed that increased P uptake can be an important component of growth stimulation by ectomycorrhizas and the first quantification of extramatrical ectomyCorrhizal hyphae in soil is presented, which suggests a role for them in the growth response.
Abstract: summary There is much circumstantial evidence for a role of increased P uptake in the growth response of plants to ectomycorrhizas. Full response curves with and without mycorrhizal infection along a P gradient in soil are, however, required to test this hypothesis fully. In this experiment, rooted cuttings of Salix viminalis L. cv. Bowles Hybrid were grown in a 1:2 mixture by volume of gamma-irradiated soil and sterile sand, with bicarbonate-extractable P concentrations of 4, 6, 10, 21, 60 or 90 mg P kg−1. The cuttings were inoculated by mixing peat/vermiculite spawn of Laccaria proxima (Boud.) Pat., or Thelephora terrestris (Ehrh.) Fr., or autoclaved spawn 1: 5 by volume with the soil: sand mixture. The plants showed a positive growth response to mycorrhizal infection by either fungus at the two lower P levels, and to L. proxima only at 10 mg P kg−1. At 21 mg kg−1and above, infection was reduced and neither mycorrhizal inoculation nor further P additions caused significant growth increases. These results imply that the growth responses to ectomycorrhizas in this experiment were solely due to increases in P uptake. Cuttings infected with L. proxima tended to be larger than those infected by T. terrestris. Estimates of percent mycorrhizal infection did not differ between the fungi at the lower P levels. However, overall production of extramatrical hyphae per gram of soil was highest in soil inoculated with L. proxima. At 10 mg P kg−1the length of L. proxima hyphae per unit length of mycorrhizal root, P uptake per unit root weight, and total P content in plants infected with L. proxima were significantly higher than for T. terrestris. This study confirms that increased P uptake can be an important component of growth stimulation by ectomycorrhizas. It also presents the first quantification of extramatrical ectomycorrhizal hyphae in soil and suggests a role for them in the growth response.

129 citations

Journal ArticleDOI
TL;DR: E ectomycorrhizas can be very effective in supplying P to their hosts even at an early stage of infection, and it is suggested that a temporal separation exists in the maximal fluxes of P and C between the fungus and the host of the myCorrhizal association.
Abstract: summary One way of viewing a mycorrhizal symbiosis is as a balance between the nutritional ‘benefits’ and carbon ‘costs’ to the phytobiont. Phosphorus acquisition efficiency (the amount of phosphorus taken up per unit of carbon allocated belowground) can be used as an indicator of this balance. In this study, phosphorus uptake and belowground carbon allocation were measured using ectomycorrhizal (M) (Thelephora terrestris (Ehrh.) Fr.) and non-mycorrhizal (NM) Salix viminalis L. cv. Bowles Hybrid. Following 50, 60, 85 or 98 d of growth in a gamma-irradiated soil/sand mixture containing 4 mg bicarbunate-extractable P kg−1, seven randomly-selected cuttings of each treatment were harvested and their P contents determined. Nine d prior to each harvest, the three median plants from the group of seven were pulse labelled with 14C to determine the relative allocation of C aboveground and belowground. Mycorrhizal colonization of willow caused a two-fold increase in growth owing to substantially higher P uptake. Phosphorus inflow rates were almost three times as high for M root systems as for NM root systems over the interval up to the first harvest (3.2 × 10 −12 and 1.2 × 1012 mol m−1 s–1, respectively). Over the interval from 50 to 98 d, inflows into M plants were 50% higher than into NM plants (1.4 × 1012 and 0.9 × 10−13 mol m−1 s−1 respectively). The M plants allocated about 25 times as much carbon belowground as the NM plants for both periods. The P acquisition efficiency was higher in M than in NM plants during the first interval (16% and 40% higher using two different calculation methods), whereas during the second interval it was higher in NM than in M plants (33% and 44% higher using the two different methods). Thus, ectomycorrhizas can be very effective in supplying P to their hosts even at an early stage of infection. Furthermore, it is suggested that a temporal separation exists in the maximal fluxes of P and C between the fungus and the host of the mycorrhizal association. The results are discussed in the context of the nutrient requirements and carbon economies of field-grown woody plants.

102 citations

Journal ArticleDOI
TL;DR: The increase in fixed carbon in mycorrhizal plants was unable to compensate for the increased carbon cost of the mycor rhizal root system, consistent with the photosynthate source-sink hypothesis.
Abstract: SUMMARY The purpose of this study was to determine how ectomycorrhizal infection and phosphorus nutrition affect biomass, photosynthesis and root respiration in the host plant. Maritime pine (Pinus pinaster Soland. in Ait.) seedlings grown in containers filled with perlite-vermiculite were inoculated with the ectomycorrhizal fungus Hebeloma cylindrosporum (strain D 3 . 25. 9) and given 0 or 0 5 mm phosphate in the nutrient solution. Hebeloma cylindrosporum infection increased net photosynthesis and root respiration rates compared with those of nonmycorrhizal plants, but there was an accompanying 35 % depression in growth. The addition of phosphorus to non-mycorrhizal plants induced a rise in tissue phosphorus content which made them similar in that respect to mycorrhizal plants but did not result in increased photosynthesis. The nitrogen content of mycorrhizal plants was, moreover, lower than that of the control group. The data recorded were consistent with the photosynthate sourcesink hypothesis. The increase in fixed carbon in mycorrhizal plants was unable to compensate for the increased carbon cost of the mycorrhizal root system.

59 citations

Frequently Asked Questions (1)
Q1. What are the contributions in this paper?

Martins et al. this paper found that mycorrhizal and nonmycizal roots accumulate orthophosphate, which indicates that this system relies upon the fungal polyphosphates as a major source of phosphate.