ABSORPTION OF PHOSPHORUS FROM SOILS BY MYGORRHIZAL PLANTS BY T. M. MORRISON

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1 ABSORPTION OF PHOSPHORUS FROM SOILS BY MYGORRHIZAL PLANTS BY T. M. MORRISON Department of Botany., University of Otago, New Zealand {Received January 19 61) (With 6 figures in the text) SUMMARY Transfer of 32p to the shoots of non-mycorrhizal and mycorrhizal plants of Pinus radiata growing in soil and in expanded perlite has been comparedby assaying the amount reaching the shoot apex. In expanded perlite and in soils with low phosphorus-sorption capacities movement of 32p to the shoot apex of non-mycorrhizal plants was rapid during an initial phase but reduced in rate during a prolonged second phase. Movement of "^^p to the shoot-tip of mycorrhizal plants, however, was at a relatively steady rate, which eventually exceeded that in non-mycorrhizal plants. In soils of high phosphorus-sorption capacity the rate of movement was reduced but followed the same course as that from expanded perlite. Transfer of 32p to the shoots of both mycorrhizal and non-mycorrhizal plants was negligible over a short period from a soil of very high phosphorus-sorption capacity. It is suggested that the apparent solubilizing effects of mycorrhizas on soil phosphorus may simply be a consequence of the rapid withdrawal of labile phosphorus by the mycorrhizal sheath. INTRODUCTION There can be little doubt that ectotrophic mycorrhizas can play an important role in the growth of coniferous species. Hatch (1937) and Mitchell et al. (1937) for instance have carried out extensive experiments using a variety of soils and demonstrated that mycorrhizas increase absorption of mineral nutrients from infertile soils. Their experiments were of necessity of long duration, 1-2 years, and their plants were analysed chemically at the end of this period. The use of ^'^P in work on mycorrhizas has enabled a more detailed examination of the uptake and internal distribution of phosphorus to be made. Kramer and Wilbur (1949) and Melin and Nilsson (1950) demonstrated that mycorrhizas accumulate more 32p in a short period of a few hours than do non-mycorrhizal roots of Pinus. In neither of these papers was an attempt made to show the distribution of the phosphorus within the mycorrhiza. Harley and McCready (1950) have obtained corresponding results with Fagus, but have also shown (1952) that 90% of the phosphorus absorbed in experiments of a few hours' duration is accumulated in the mantle of the mycorrhiza. Later, however, they were able to demonstrate a gradual transfer of phosphorus from the mantle to the host core and postulated (1955) that it was potentially capable of transferring substantial amounts of phosphorus to the host tissues. In the duration of their experiments, however, the amount of phosphorus absorbed by non-mycorrhizal roots was generally double the amount of phosphorus transferred from the mantle to the host tissues in mycorrhizas

2 Phosphorus absorption 11 (1952). This apparent contradiction in results obtained by using '^-P for short periods and those of earlier workers using unlabelled phosphorus for long periods 'complicates the simple view of mycorrhizas as highly efficient salt-absorbing organs' (Harley, 1956). This contradiction appears, however, to be a consequence of the short duration of the ^sp experiments. Work with intact plants whose roots were supplied with ^sp has since shown (Morrison, 1957) that while shoots of non-mycorrhizal plants initially receive labelled phosphorus at a rapid rate this is followed by a second period of slow movement to the tip. Shoot tips of mycorrhizal plants under the same conditions receive phosphorus at a relatively constant rate. This rate is less than that of the initial period but greater than that of the second period in shoots of non-mycorrhizal plants. Furthermore, the phosphorus status of shoots of non-mycorrhizal plants afitects the rate at which they receive labelled phosphorus, but has little effect in mycorrhizal plants at least during the first few days after application of labelled phosphorus. However, the rate of arrival of 3-P at the shoot tips of mycorrhizal and non-mycorrhizal pines has hitherto been compared only in expanded perlite* cultures. The first experiment described in this paper confirms and extends the results obtained in this medium. Subsequently the shapes of the uptake curves are compared in shoot-tips of plants grown in soils differing in their phosphorus-sorption capacities. METHODS AND RESULTS Experiment i. Uptake of ^-P from expanded perlite cultures This experiment was carried out in the same manner as that previously reported (Morrison, 1957). Seeds of Piniis radiata were sterilized by immersion in a solution of calcium hypochlorite (Morrison, 1957b). They were then sown in expanded perlite without any nutrient additions. As soon as possible after germination seedlings were pricked out into a mixture of sterilized expanded perlite inoculated with % of pine humus to produce mycorrhizal plants or with sterilized humus to produce non-mycorrhizal plants. Thirteen weeks after pricking out, the seedlings were transferred to individual glass pots of 500 ml capacity containing expanded perlite. During this pretreatment period plants were divided into four blocks. Two main blocks mycorrhizal and non-mycorrhizal were each subdivided into two groups those receiving a full nutrient solution (HP) and those receiving a nutrient solution lacking phosphorus (LP). Solutions were prepared with distilled water and had an initial ph of approximately 5. Fresh solution was added every 14 days and drainage, supplemented if necessary with distilled water, was returned to each pot, so that pots were fully saturated three times weekly. All pots received the following solution (g/1): KNO , H3BO , CuCl , Ca(NO3)2.4H2O 0.944, MnCl2.4H2O , Molybdic acid , MgSO4.7H2O 0.369, ZnCl , Fe-EDTA In addition pots in the treatment HP received NaH2PO4.2H2O at the rate of g/1. of solution while pots in the treatment LP received g NaCl per litre of solution to provide sodium ions. Plants were grown during this period in a heated glasshouse with supplementary lighting. After 17 weeks of this pretreatment ten representative plants from each of the four blocks were transferred to individual experimental pots containing expanded perlite. During this transfer roots were washed thoroughly with tapwater and examined for the presence or absence of mycorrhizas. The following nutrients M^ere then supplied to each * An inert material from volcanic glass and kindly supplied by Giles & Elliot Ltd., Wellington, N.Z.

3 12 T. M. MORRISON pot (mg/pot): KNO3 20, H3BO3 0.29, CuCl , Ca(NO3)2.2H2O , Molybdic acid 0.002, MgSO4.7H2O 37, ZnCb 0.012, Fe-EDTA 5, Phosphorus was labelled by addition of radioactive H332PO4 to the nutrient solution so that each pot received 100MC of ^-F. Thereafter drainage was returned frequently, after being made up if necessary with distilled water to sufficient volume to saturate each pot. O O Days Fig. I. Growth of mycorrhizal ( ) and non-mycorrhizal (. _) plants in expanded perlite as measured by stem height. LP, lacking phosphorus; HP, supplying a normal level of phosphorus. Eighteen plants per treatment. Shoot-tips of all plants were assayed for activity daily as previously described (Morrison, 1957). On the eleventh day after addition of ^ap some plants from each treatment were given additional treatments (not discussed here) so that the number of replicates in the main blocks was reduced from ten to five from this date. On the twenty-fourth day after addition of ^ap all plants received nutrient solution of the same composition as previously except that the activity of 32p had decayed from 100 nc to 31 ^c/pot. On the fifty-second day after addition of 32p plants from the pretreatment LP (low phosphorus) received a further application of nutrient solution in which 8 MC of 32p was added per pot. In the presentation of the results allowance was made for decay of the isotope according to published tables (Wright, 1958) and probe corrections were made for actual counts exceeding 7000 per minute. 120

4 Phosphorus absorption 13 Growth of plants during the 17 weeks' pretreatment period as measured by stem increments is shown in Fig. i. This was much affected by the level of phosphorus in the nutrient solution, but no clear effect of mycorrhizas on growth was recorded. This was not unexpected since in one treatment (HP) a well-balanced supply of nutrients was available while the other (LP) lacked phosphorus entirely. Fig. 2. Activity of shoot-tips of mycorrhizal ( ) and non-mycorrhizal ( : : ) plants of low phosphorus status (LP) growing in expanded perlite after addition of ^"P. Ten plants per treatment from o to ii days, thereafter five plants per treatment. \'crtical lines represent dates on which fresh solution was added. Arrival of 32p at shoot-tips of plants exposed to this element is shown in Figs. 2 and 3. These results substantiate those already published (Morrison, 1957) in that arrival of phosphorus at shoot-tips of non-mycorrhizal plants after addition of 32p consisted of two clear phases a period of rapid movement and a second period of less rapid movement whatever the phosphorus status of the plants. This pattern re-occurred each time that the solution was replenished. On the other hand arrival of 32p at shoot-tips of mycorrhizal plants consisted of a single phase and was largely unaffected by replenishment Days

5 T. M. MORRISON of the solution. During the first week of ^ap uptake the phosphorus status of mycorrhizal plants had little effect on the amount of ^ap reaching the shoot-tips but in nonmycorrhizal plants a depressing effect of an initially high phosphorus content was immediately obvious. In the course of the experiment plants in the LP treatments were progressively brought closer in total phosphorus content to plants in the HP treatments and there is an increasingly close correspondence in the shape of the curves for nonmycorrhizal plants after the second and third additions of 3O Q Days Fig. 3. Activity of shoot-tips of mycorrhizal ( ) and non-mycorrhizal (O O) plants of high phosphorus status (HP) growing in expanded perlite after addition of ^ap. Ten plants per treatment from o to 11 days, thereafter five plants per treatment. Vertical lines represent dates on which fresh solution was added. As a consequence of this pattern of uptake and movement, 2 days after addition of the radioactive element shoot-tips of non-mycorrhizal plants had received at least thirteen times more ^'^P than had shoot-tips of mycorrhizal plants of similarly low phosphorus status. Despite this large disparity in initial rates, radioactivity of shoot-tips of mycorrhizal plants of low phosphorus status had exceeded that of non-mycorrhizal plants by the eightieth day of the experiment. Results obtained from plants of high phosphorus status were inconclusive in this experiment in that although the rate of movement of 32p [^ mycorrhizal plants approached and finally exceeded that in non-mycorrhizal plants the total activity of the former did not exceed that of the latter when the experiment was terminated. Experiment 2. Uptake of ^^P from a soil of high phosphorus-fixing 4O capacity The soil of volcanic origin, Taupo Hill soil (N.Z. Soil Bureau, 1954) used in this experiment had the following analysis (per cent dry weight.): ph Available P2O,5 Total N C.E.C. T.E.B. Base saturation 5-8 Ca 6.6 m.e % Mg i.o m.e. / 0.48'% K 0.35 m.e. % 17.5 m.e. % Na 0.4 m.e. % 8.1 m.e. % 46% 5O

6 Phosphorus absorption The soil was mixed with an equal volume of washed silica sand, autoclaved, potted and fertilized some months before seedlings were transplanted to it. The experiment consisted of two blocks - mycorrhizal and non-mycorrhizal - each of which was subdivided into two treatments: (i) receiving no supplementary nutrients (o), (2) receiving a full nutrient dressing including phosphorus (P): NH4NO3 0.5 g/pot Na2B4O7,ioH K2SO4 0.5 g/pot ZnSO4.7H2O 0.07 g/pot CUSO4.5H2O 0,5 g/pot each g/pot The fertilizer was added to each pot in 200 ml of solution. Pots were of glass and held 529 g (oven-dry weight) of the medium. Treatments were replicated eight times. Mycorrhizal and non-mycorrhizal seedhngs of P. radiata were raised as described in Experiment i but were transferred to the experimental pots 10 weeks after germination. ir 2r-..--' ^O"" ^ o- O OP Da ys Fig, 4, Activity of shoot-tips of mycorrhizal ( ) and non-mycorrhizal (O (j) plants after addition of ''^P, Plants were growing in a soil of high phosphorus-sorption capacity which had either received no fertilizers (O) or had received fertilizers including phosphorus (P), Six plants per treatment. Plants remained in these pots for 8 weeks when four from each treatment were harvested, dried, weighed and analysed for total phosphorus. The following day all remaining pots received 150 ml of a solution containing 50MC ^^p gg H3PO4 and io^^m. NaH2PO4,2H2O as a carrier. Daily activity measurements were made on apices of all plants by the method previously described. The experiment was dismantled 21 days after application of ^^P. Table i. Total phosphorus [mg P/100 g. dry weight) in shoots of plants before addition of ^^-P. {Means of four plants) Treatment o Myc, Nonmyc, Myc, Nonmyc, Dry weight (g) 0,226 0,211 0,327 0,252 mgp per 100 g dry weight s

7 i6 T. M. MORRISON Growth and total phosphorus in the shoots were both increased by the presence of mycorrhizas and by addition of nutrients to the soil (Table i). In Fig. 4 daily activities are shown of shoot-tips of plants exposed to -^-P for 2i days. Pre-addition of fertilizer substantially increased 32p movement to the shoots of both mycorrhizal and non-mycorrhizal plants. In the absence of fertilizer, activity of shoottips was barely measurable by the method employed. In media which had been prefertilized, shoots of non-mycorrhizal plants showed an initial phase of rapid movement of 32P lasting about io days followed by a second phase of less rapid but relatively steady movement. Shoots of mycorrhizal plants on the other hand had a constant rate of 32p movement to the tips throughout the experiment, less than that of the initial period but greater than that of the second period in non-mycorrhizal plants. This course of uptake IS similar to that described for plants in expanded perlite. - -O 0 a y s Fig. s. Activity of shoot-tips of mycorrhizal ( ) and non-mycorrhizal (C growing in fertile soil mixtures after addition of ''^P. Four plants per treatment. O) plants Experiment 3. Uptake of ^'-P from soil mixtures The media used in this experiment consisted of loam-sand (L) and loam-peat-sand (P) mixtures with the following composition by weight: L Pa Pb Loam Peat 2-5 O-5 Sand The loam was an alluvial sandy loam, Clutha fine sandy loam (Cutler, 1957) with ph 6.0, base exchange capacity of 8 m.e. % and a base saturation of %. The peat had a ph of 2.5, base exchange capacity of 117 m.e. % and a base saturation of 16 %. The sand had been washed with tap-water, autoclaved and oven dried before mixing with the chloropicrin-sterilized loam and peat. Clay used in the media was kaolinite with a base exchange capacity of 12 m.e. % and had been acid treated to remove bases. Media were Clay

8 Phosphorus absorption 17 contained in glass pots as in the previous experiments. There were four replicates of each treatment. Mycorrhizal and non-mycorrhizal plants of P. radiata, raised as previously described, were transplanted into the experimental pots 4 months after germination and 41 uc of 32P was added to each pot 4^ months later. Daily activity measurements were made on all plants as in the previous experiments for the next 14 days. In media L and Pa changes in activity of the shoot tips during this 14-day period are shown in Fig. 5 these were similar to those recorded in Fxperiment 2. Thus in nonmycorrhizal plants a relatively rapid initial movement of 32p was followed by a second period of less rapid movement while in mycorrhizal plants the rate of movement was constant throughout the experiment....-o o,-' / 'o 0/ Days Fig. 6. Activity of shoot-tips of mycorrhizal ( ) and non-mycorrhizal (O O) plantsafter addition of ^^P. Plants were growing m an infertile soil mixture. Four plants per treatment. The arrival of ^^P at the shoot-tips of phosphorus-starved plants in Pb is shown in Fig. 6. Movement of ^^P followed substantially the same course as that shown in the first 14 days of uptake by plants of low P status in Fxperiment i. Thus the initial movement in non-mycorrhizal plants was extremely rapid and the same clear division into first and second phases was apparent. Movement of ^^P in mycorrhizal plants was, however, slow in comparison but followed a straight-line course with time. B N.P.

9 i8 T. M. MORRISON DISCUSSION The rate of movement of phosphorus 32p to the shoot-tips of plants depends on several factors: (1) rate of uptake by the roots, (2) availability of exchange sites in the plant whether these be an exchangeable-phosphorus pool or immobile accumulation sites (3) rate of translocation in the plant. In comparing mycorrhizal and non-mycorrhizal plants some of the above factors can be dismissed. There is no recorded evidence that the rate of movement of solutes is different in mycorrhizal and non-mycorrhizal plants nor is there any evidence to suggest that exchange sites are dissimilar in the host tissues of a mycorrhizal plant and a nonmycorrhizal plant. In fact it is in the rate of uptake by the roots and the presence of exchange and accumulation sites in the fungal component of mycorrhizas that the plants differ. The presence of exchange and accumulation sites in mycorrhizas probably prevents the initial rapid movement of phosphorus to the shoot-tips of these plants although in plants of lov^' phosphorus status the exchange sites at the tip will be largely unsaturated. That there is an initial large storage of phosphorus in the fungus mantle was shown indirectly previously (Morrison, 1957) by transferring plants after 6 days in 32p in solution to phosphorus-free media shoots of mycorrhizal plants continued to increase in activity by 2000 counts/minute while non-mycorrhizal plants increased by 400 counts/ minute. This increase in activity of mycorrhizal plants probably represented 32p that had been stored in the fungus mantle. These results are in accord with those reported by Harley et al. who demonstrated directly a rapid accumulation of ^^p in the mantle of beech mycorrhizas. The constant transfer of phosphorus to the host from the fungus that begins at the same time as movement of '^'^p through a non-mycorrhizal plant and, at least in the early period of absorption, its independence of phosphorus status of the shoots, suggests that there is a constant leakage of phosphorus to the host from the fungus. It is shown in these experiments that if phosphorus in the medium is readily available the rate of movement of ^sp to the shoot tips after the period of initial exchange is over, is greater in mycorrhizal than in non-mycorrhizal plants. The conclusion is therefore inescapable that plants possessing mycorrhizas have a faster rate of uptake of phosphorus than those lacking mycorrhizas. In soil, phosphorus is not always readily available to plants. Thus, in Experiment 2, plants grown in soil with a high capacity for phosphorus sorption and which had received no pretreatment with phosphorus, failed to exhibit any appreciable uptake of ^^p applied to the soil whereas plants grown in this soil which had previously received a liberal dressing of phosphorus, showed a substantial uptake of ^''P despite the higher phosphorus status of the plants. After addition of 32p leachates from pots containing this soil and previously receiving phosphorus fertilizer had no discerniljle activity. Schofield (1955) regards phosphorus in soil as consisting of a labile pool in equilibrium with a solid phase. The labile pool consists of phosphorus in the soil solution and phosphorus held on exchange sites in the soil complex the former ions having a higher potential or free energy than the latter. Russell et al. (1958) have pointed out that 'absorption of phosphorus by plants must depend on root surfaces reducing the free energy of the entering ions to a greater extent than complexes' in the soil and they have shown that inter-

10 Phosphorits absorption 19 specific differences in the ability of plants to lower the free energy of phosphorus do exist. Stone (1949) has demonstrated that mycorrhizas of P, radiata cannot dissolve insoluble phosphorus any better than non-mycorrhizal roots but can render 'fixed' soil phosphorus available to the host but not to other plant roots growing in the same pot. For this the mechanism is now apparent. It is evident that mycorrhizas are able to lower the free energy of phosphorus more than non-mycorrhizal roots and the mechanism would appear to be a more rapid utilization of phosphorus by mycorrhizas than occurs in nonmycorrhizal roots. It is thus unnecessary to claim (e.g, Routien and Dawson, 1943) that mycorrhizal fungi exude phosphate-solubilizing substances and thus enhance phosphorus uptake from soils. They need merely withdraw labile phosphorus from the medium more rapidly than do the uninfected roots of their host. Quahtatively, uptake is similar from soil and expanded perlite but in the former uptake can be prevented by phosphorus sorption in the medium. Thus if the phosphorussorption capacity of the medium is lowered either by dilution of the soil with large quantities of sand (Treatment Pb, Experiment 3) or by previous addition of phosphorus (Experiment 2), uptake of 32p is quantitatively similar to that from expanded perlite culture. However, if the phosphorus-sorption capacity is very high while the total amount of phosphorus added is low, plants whether mycorrhizal or not fail to translocate appreciable quantities of added 32p to the shoot-tip during short periods of 21 days. In treatment Pb of Experiment 3, plants had been growing in a medium of extreme infertility and would therefore be of low phosphorus status. This medium did not appreciably sorb phosphorus since high counts were recorded from leaehates after addition of ^^F. In similar circumstances it was shown previously (Morrison, 1957), and in Experiment i, that non-mycorrhizal plants had an extremely rapid initial uptake of phosphorus while uptake by mycorrhizal plants was unaffected by their phosphorus status during the first few days after adding ^-P, A plant carrying ectotrophic mycorrhizas can be regarded as buffered from the soil by the fungal weft on its roots so that it is relatively insensitive to changes in its rooting environment. Thus in Experiment i, mycorrhizal plants showed little change in the rate at which ^~P reached the shoot tips when the solution was replenished, and it was shown previously (Morrison, 1957) that this rate was substantially unaltered over a period of 15 days after mycorrhizal plants were removed to a phosphorus-free medium. How far this holds for other mineral nutrients and water absorbed by mycorrhizas is not known but Kramer (1956) has pointed out the possibility that mycorrhizas play a part in water absorption. ACKNOWLEDGMENTS Part of this work was carried out at the Soil Bureau, D.S.I.R., Wellington, N.Z. The remainder was financed by grants from the University of New Zealand, I am indebted to Dr. R. Scott Russell and Dr. J. L. Harley for critical discussions of this material. REFERENCES CUTLER, E, (1957), Soils of Lower Clutha Plain. N.Z. Soil Bur. Bull., n,s. 15, HARLEY, J, L, & MCCREADY, C, C, (1950), The uptake of phosphate by excised mycorrhizal roots of the heech. Nezu PhytoL, 49, 38S.,.,, u J U-, CU HARLEY, J, L, & MCCREADY, C, C, (1952), The uptake of phosphate by excised mycorrhizal roots of the beech. H. Distribution of phosphorus between host and fungus. Nezv PhytoL, 51, 342,

11 2O T. M. MORRISON HARLEY, J. L. & BRIERLEY, J. K. (1954). The uptake of phosphate by excised mycorrhizal roots of the beech. VI. Active transport of phosphorus from the fungal sheath into the host tissue. New Phytol., r u u HARLEY, J. L. & BRIERLEY, J. K. (1955). The uptake of phosphate by excised mycorrhizal roots ot the beech. VII. Active transport of -'-P from fungus to host during uptake of phosphate from solution. Neio Phytol., 54, 296. HARLEY, J. L. (1956). The mycorrhiza of forest trees. Endeavour, 15, 43. HATCH, A. B. (1937). The physical basis of mycotrophy in Pinus. Black Rock For. Bull., No. 6. KRAMER, P. J. & WILBUR, K. M. (1949). Absorption of radioactive phosphorus by mycorrhizal roots of pine. Science, no, 8. MELIN, E. & NiLSSON, H. (1950). Transfer of radioactive phosphorus to pine seedlings by means of mycorrhizal hyphae. Physiol Plant., 3, 88. MITCHELL, H. L., FINN, R. F. & ROSENDAHL, R. O. (1937). The relation between mycorrhizae and the growth and nutrient absorption of coniferous seedlings in nursery beds. Black Rock For. Papers, I, 58. MORRISON, T. M. (19S4). Uptake of phosphorus-32 by mycorrhizal plants. Nature, 174, 606. MORRISON, T. M. (1957). Mycorrhiza and phosphorus uptake. Nature, 179, 907. MORRISON, T. M. (1957b). Host-endophyte relationships in mycorrhizas of Pernettya macrostigma. New Phytol., 56, 247. N.Z. SOIL BUREAU (1954). General Survey of the soils of North Island, New Zealand. N.Z. Soil Bur. Bull., " ^- 5- RouTiEN, J. B. & DAVVSON, R. F. (1943). Some inter-relationships of growth, salt absorption, respiration and mycorrhizal development in Pinus echinata. Amer.J. Bot., 30, 440. RUSSELL, R. SCOTT, RUSSELL, E. W. & MARAIS, P. G. (1958). Factors affecting the ability of plants to absorb phosphate from soil. II. A comparison of the ability of different species to absorb labile soil phosphate. Soil Science, 9, ioi. SCHOFIELD, R. K. (1955). Can a precise meaning be given to 'available' soil phosphorus? Soils and Fert., 18, 373- STONE, E. L. (1949). Some effects of mycorrhizae on the phosphorus nutrition of Monterey pine seedlings. Soil Set. Soc. Amer. Proc, 14, 340. WRIGHT, E. (1957). Some decay tables for use with radioactive tracers. N.Z.J. Sci. Tech. A 38, 1091.

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