AUTORADIOGRAPHY OF THE DEPLETION ZONE OF PHOSPHATE AROUND ONION ROOTS IN THE PRESENCE OF VESICULAR-ARBUSCULAR MYCORRHIZA

New Phytol. (1979) 82, 133-140 AUTORADIOGRAPHY OF THE DEPLETION ZONE OF PHOSPHATE AROUND ONION ROOTS IN THE PRESENCE OF VESICULAR-ARBUSCULAR MYCORRHIZA BY E. OWUSU-BENNOAH AND A. WILD Department of Soil Sciencey The University^ Reading RGl 5AQy U.K. {Accepted 4 July 1978) SUMMARY Using autoradiography a comparison was made of the depletion zones of ^^P-Iabelled phosphate around mycorrhizal {Glomus mosseae) and non-mycorrhizal onion roots grown in a silty clay loam for 42 days. Mycorrhizal inoculation increased the growth and ^^P content of the onion plants in both irradiated and non-irradiated soil. Successive autoradiographs showed that for the mycorrhizal and non-mycorrhizal roots most P uptake was within 0-2 and 0-1 cm respectively of the root surface. The effect of the mycorrhiza was similar to that of a cylinder of root hairs 0-1 cm long. INTRODUCTION Infection with vesicular-arbuscular (VA) mycorrhizal fungi increases the growth of temperate and tropical crop plants, the effect usually being due to increased phosphate uptake (Mosse, 1973; Gerdemann, 1975; Tinker, 1975; Powell and Daniel, 1978). The usual explanation for this greater uptake of phosphate is that hyphae which grow away from the roots absorb phosphate which is transferred to the host, thereby increasing the volume of soil which the roots exploit (Gerdemann, 1968; Hayman and Mosse, 1971; Sanders and Tinker, 1973). It has been estimated that per cm of infected root there are 80 cm of hyphae (Sanders and Tinker, 1973) making about 46 to 107 connexions with the root (Mosse, 1959). Although some hyphae have been reported to grow 1 to 7 cm from the host root (Hattingh, Gray & Gerdemann, 1973; Pearson and Tinker, 1975; Rhodes and Gerdemann, 1975) their effectiveness in absorbing phosphate will depend on their population density. The objective of our work was to use the size of the depletion zone of ^^P-labelled phosphate as a measure of the distribution of hyphae. For this purpose the depletion zones were measured close to mycorrhizal and non-mycorrhizal roots. MATERIALS AND METHODS Soil A silty clay loam, Hamble Series (Jarvis, 1968), was sampled at a depth of 5 to 20 cm at a site near Reading (grid ref. SU 616703) after a wheat crop. It was first cropped with Lolium perenne in pots to reduce the size of the pool of labile phosphate. During a period of 30 weeks, 14 cuts of the grass removed 66 mg P per kg soil. After air-drying and removal of roots part was crushed to pass a 100-mesh (0-152 mm) 0028-646X/79/0101-0133 $02.00/0 1979 The New Phytologist

134 E. OWUSU-BENNOAH AND A. WILD Table 1. Physical and chemical properties of the Ha7tible soil Texture Organic matter (%) ph in 0-01 M-CaCU CaCla-sol P ifimo] 1"^)* 0-5 M-NaHCOg-sol P (mg kg-^)t Resin-P (mgkg~^)!l; E-value (mg kg~'^) Phosphate buffer capacity SUty clay loam 1-64 7-3 054 19-8 33-5 32-1 3-5x102 * 1 g soil shaken 24 h in 20 cm^ 001 t 1 g soil shaken 30 min m 20 cm^ NaHCOg at ph 8-5. jig soil shaken 24 h in 20 ml water with addition of 2 g Zeroht FF anion exchange resin (Cl'). 1-5 g soil shaken 24 h in 29 cm* 0-01 M-CaCU, suspension shaken further 24 h after addition of 2-5 /tci 32p (1 cm^) and toluene. II Using the resin method of Brewster et al. (1975). SecHon locm I unlabelied 2 cm H labelled 37 cm :^ HI uniabelled 4 cm Fig. 1. Diagrammatic representation of soil block. sieve. The ph, percentage organic matter and the phosphorus status of the phosphatedepleted sample are given in Table 1. Apparatus The containers used to grow the plants were similar to those of Bhat and Nye (1973). Each contained a block of soil 49 cm long, 4 cm wide and 0-8 cm deep. In sections I and III of each block (Fig. 1) unlabelied soil (< 2 mm) was packed at a dry bulk density of 1-1. Section II contained fine soil (< 0-152 mm) at the same bulk density labelled with carrier-free H3 ^^P04 at 5*5 /icig~^ soil. Eine soil was used in Section II in order to give an autoradiograph with a uniform background. Water was added to each block to give an average content of 0*35 cni^ cm~^, which was

Depletion of phosphate around mycorrhizal roots 135 maintained constant during plant growth. The weight of each block was checked twice daily, and when necessary additional water was sprinkled on the surface. Eight containers were used for four treatments in duplicate. The treatments were: soil irradiation (*sterilization*) with 0-8 Mrad of y-radiation given prior to the preparation of the soil blocks and no irradiation, each with and without mycorrhizal inoculation. The treatments will be referred to as S + (sterilized plus inoculum), S (sterilized without inoculum), N+ (non-sterile plus inoculum), N (non-sterile without inoculum). Culture of onion plants Onion seedlings {Allium cepa var. White Lisbon) germinated on filter paper until the radicles were 1 cm long, were transferred to small holes made in a 5 mm thick layer of candle wax resting on top of each soil block. The radicle extended into the soil but the seed was held in the wax to minimize the number of seminal roots. Two seedlings were placed against the inner face of the front plate of the cell, which was tilted to induce the root to grow down the surface of the block. The cells were placed in a growth cabinet and given a 16 h day, with night and day temperatures of 16 and 24 C respectively. Inoculation In the S+ and N+ treatments the plants were inoculated with Endogone YV (yellow vacuolate) spore type (Mosse and Bowen, 1968), named Glomus mosseae by Gerdemann and Trappe (1974). It had been maintained in pot culture of Nardus stricta. The inoculum consisted of sporocarps, spores, infected root fragments and mycelium which had been separated by wet-sieving (< 106/*m mesh) and decanting (Gerdemann and Nicholson, 1963). A band of soil 5-0 mm wide was carefully removed from Sections I and III of each soil block 1-0 cm above and below Section II. The spaces created were packed with the inoculum. A further 10 g of the inoculum was mixed with 50 ml of deionized water, and 2 cm^ of this inoculum was sprayed on the roots during their grovith through the soil in Section I of the block. The roots in treatments S and N were sprayed with 2-0 cm^ of the filtrate from the inoculum, which was free of Endogone mycorrhiza but contained contaminating micro-organisms. A utoradiography Twenty-one days after planting, the roots had grown into the ^^P-labelled section of the soil block. The front plate of the cell was removed and the soil surface was covered in turn with a 3 /im thick mylar film and Kodirex X-ray film. To ensure good contact between the X-ray film and the soil surface, a perspex block weighting 50 g was placed on top of the film. After 4 h exposure the film was developed, fixed and dried. Autoradiographs were taken every 7 days thereafter until day 42 when the experiment was terminated, the exposure time being increased to allow for the decay of the radio-isotope. At the same times, autoradiographs were also taken of a range of standards prepared by adding ^^P to the same soil. All autoradiographs were scanned by a Wooster Mark III recording microdensitometer using a scan magnification of x 5. Densities were measured at 0-5 cm intervals within Section II of the blocks and were converted into ^^P concentrations (c.p.m.) by reference to the standards.

136 E. OWUSU-BENNOAH AND A. WILD Analyses After 42 days of growth the section of the roots which grew into the 2-0 cm labelled soil band were carefully removed, washed and the diameter measured under a microscope using a graduated eye-piece graticule. They were cleared and stained by the method of Phillips and Hayman (1970). The stained roots were examined under the microscope for mycorrhizal infection. The shoots and those parts of the roots not used for staining, were washed, dried and weighed. The shoots and roots were separately analysed for phosphate by the molybdenum blue method after digesting the plant material in nitric/perchloric/ sulphuric acids (Jackson, 1966). The ^^P content of the plants was determined by liquid scintillation counting of the digest using Triton X-100 as scintillant. RESULTS Growth and phosphate uptake Inoculation with the endophyte gave greater yields of plant material whether or not the soil was irradiated. Dry weights of shoots were greater with the mycorrhizal Table 2. Effect of VA mycorrhiza on onion grown in irradiated and non-sterile Hamble soil for 42 days (means of two soil blocks) {weights as g per plant) Treatment Root (cm) weight Root Dry weight * Shoot ratio Irradiated sou- -mycorrhiza Irradiated soil mycorrhiza Non-sterile soil + mycorrhiza Non sterile soil - mycorrhiza L.S.D. 0-08 006 0-06 004 1-85* 1-05'^ 1-35*' 1-07'^ 0-46 0-04* 0-03* 0-04* 0-03* 0-02 0-16' 0-06'* 0-09'' o-os'' 0-06 0-25 0-50 0-44 0-60 0-23 In each column, values followed by the same letter are not significantly different (P = 0-05). Table 3. The effect of VA mycorrhiza on the absorption of phosphate {minus seed P) by onion plants grozvn for 42 days in irradiated and non-irradiated Hamble soil {means of two soil blocks) P concen- P uptake (mg plant"^) Treatment (%) Root Shoot Total Irradiated 4- mycorrhiza Irradiated mycorrhiza Non-sterile - - mvcorrhiza Non-sterile mycorrhiza L.S.D. 0-24" 0-14^ 0-21* 0-14'' 0-05 0-07* 0-05" oos^ 0-04'» 0-02 0-40* 0-08'' 0-22*= 0-07'> 0-06 0-47* 0-13'' 0-27'' O-U*" 005 In each column, values followed by the same letter are not significantly different (P = 0-05). plants than with the non-mycorrhizal plants, and as the root dry weights were not affected the mycorrhizal plants had lower root: shoot ratios than the controls (Table 2). Examination of the stained 2-0 cm segments of roots which grew into the labelled soil showed that the inoculated roots were heavily infected. It was not possible to quantify the degree of mycorrhizal infection because of the small sample examined.

Depletion of phosphate around mycorrhizal roots 137 However, abundant external and internal hyphae were visible in all the inoculated roots, whereas the non-inoculated plants remained non-mycorrhizal even in the nonsterile soil. The mycorrhizal plants had a higher percentage P in their dry matter than nonmycorrhizal plants (Table 3), which together with the higher dry-matter production gave higher amounts of phosphate uptake. The uptake of ^^P showed the same trends (Table 4). Table 4. Uptake of ^^P by miian plants grown with and without mycorrhiza for 42 days, and depletion in soil {ctjmin per plant) Treatment Irradiated + mycorrhiza Irradiated mycorrhiza Non-sterile + mycorrhiza Non-sterile mycorrhiza Root 4003 2814 2914 2349 Uptake by plant Shoot 10188 4576 5999 4168 Total 14191 7390 8913 6517 > Depletion in soil 11313 7138 8 543 6031 Distance (cml 2 Fig. 2. Densitometer scans ( x 5) across autoradiographs of (a) mycorrhizal (S 4-) and (b) nonmycorrhizal (S ) roots, in the centre of the labelled soil (Section II); root marked with broken line. Depletion patterns around mycorrhizal and non-mycorrhizal onion roots Figure 2 gives examples of densitometer scans across the autoradiographs of mycorrhizal and non-mycorrhizal roots 1-0 cm from the surface of the labelled band at the end of the experiment. They show a fairly uniform baseline, indicating that the ^^P had been evenly distributed in the soil. The amounts of ^T removed from the soil by mycorrhizal and non-mycorrhizal roots were calculated from the microdensitometer

138 E. OWUSU-BENNOAH AND A. WILD scans at varying distances (0-02 cm apart) from the root surface (Bhat and Nye 1973). Figure 3(a) and (b) shows that similar amounts of -^^P had been removed by mycorrhizal and non-mycorrhizal plants 9 and 16 days after the mycorrhizal roots had grown through the inoculum, which correspond to periods of 21 and 28 days after planting. By 23 days [Fig. 3(c)] the volume of soil depleted in ^^P by the mycorrhizal plants had become greater, whereas there had been little change round the nonmycorrhizal roots. Similar results were obtained at the end of the experiment, with 100.0) b} 80 ^soil) 60 o 40 removed from soil: (cpm m 20 200 160 120 {c (d 80 40 001 005 009 O'OI 0 05 0 09 013 017 Distance from roof surface (cm) Fig. 3. Depletion of "P in soil as a function of distance from root surface, based on scans across autoradiographs in centre of the labelled soil (Section II). The periods after the root had grown through the zone of the inoculum are 10, 17, 24, 31 days respectively in the four successive parts of the figure, (a) 21 days after planting; (b) 28 days after planting; (c) 35 days after planting; (d) 42 days after planting. A, N+; D, N-; O, S-1-; A, S-. more extensive depletion around the mycorrhizal than the non-mycorrhizal roots [Fig. 3(d)]. The results show that the mycorrhizal roots in the irradiated soil removed a greater amount of ^^p close to the root than the control plants, and the depletion zone spreads out more from the root surface. From the buffer capacity of the soil (Table 1), a volumetric water content of 0-35 and a measured impedance factor of 0-30, the diffusion coefficient of phosphate in the soil was 3x10"^ cm^ s"^ The radius of the depletion zone is given approximately by ^JDt (where D = diffusion coefficient and t = time), and for 23 days this gives a value for the non-mycorrhizal roots of 0-08 cm, which is in reasonably good agreement with the measured value of 0-1 cm [Fig. 3(c)],

Depletio7i of phosphate around mycorrhizal roots 139 DISCUSSION Root infection by the mycorrhiza was not investigated during growth in order to avoid damage to the plants. However, 9 and 16 days after the roots had grown through the inoculum the ^^P depletion zones of the mycorrhizal and non-mycorrhizal plants were similar in size. It therefore seems unlikely that infection occurred by 16 days. Subsequently, there was little change in the extension of the depletion zone around nonmycorrhizal plants, and at harvest the plants were smaller and the uptake of ^^P and ^^P were less than from the mycorrhizal plants. The change in the depletion zones at 23 days around the mycorrhizal plants but not the controls supports the evidence that mycorrhizal infection starts to function actively about 21 days after inoculation (Gerdemann, 1968; Mosse, Hayman and Ide, 1969; Sanders and Tinker, 1973). Figure 3(c), (d) indicates that the increased P uptake (Tables 3 and 4) by the mycorrhizal plants grown in sterilized soil occurred within 0-2 cm of the root surface, and the effect of the mycorrhiza is to increase the radius of the depletion zone from about 0-1 to 0-2 cm. Since the growth of hyphae into the soil block (S + ) is likely to be in all directions from any particular entry point on the root, it is reasonable to assume that the fungus would explore a cylindrical volume of soil around the root. By analogy with the root hair cylinder model, P depletion from the mycorrhizal zone was calculated using the formula (Nye, 1966): P depletion = \7T{al-a;) ACly where / is the length of root section considered, 0-5 cm; a^ is the radius of the ' mycorrhizal cylinder ^; a^is the radius of the primary root section (0-037 cm); A C is the drop in concentration of exchangeable P within the mycorrhizal cylinder in ct/min. Assuming a value for a^ of 0*1 cm, the calculated P depletion from within the mycorrhizal cylinder at the end of the experiment in S+ was 3432 ct/min cm~^. The total depletion was 11313 (Table 4), giving a difference of 7881 ct/min. This is similar to the depletion of ^^P around the non-mycorrhizal roots (Table 4), suggesting that most of the hyphae are within about 0-1 cm of the root surface. The effect of the hyphae in extending the radius of the phosphate depletion zone from about 0-1 cm to 0*2 cm is similar to the effect of root hairs (Lewis and Quirk, 1967; Bhat and Nye, 1974; Temple-Smith and Menary, 1977), These observations differ from those of Hattingh et al. (1973), who showed that mycorrhizal hyphae extend at least 2-7 cm from the root surface. Rhodes and Gerdemann (1975) also used a modified soil chamber to show that mycorrhizal hyphae can translocate ^^P from distances of up to about 7 cm from the root surface. The conditions on the soil plane may, however, have induced more profuse hyphal growth than is possible under normal field conditions (Hattingh, 1975). In the present experiments the finely crushed soil in Section II contained small pore spaces which might have been nearly water-saturated, and this may have restricted the growth of the hyphae. A limitation to the present autoradiographic technique is that it is not able to detect changes of P concentration of less than 10 to 20%. With the mycorrhizal plants in sterile soils (S + ) the estimate of ^ **P depletion was 20% less than measured ^^P uptake, although with other treatments the agreement was close (Table 4). Thus, although there might have been a small amount of ^^P taken up by the hyphae at a

140 E. OWUSU-BENNOAH AND A. WILD greater distance from the root surface, the main increase of phosphate uptake due to the presence of the endophyte was from soil within 0-2 cm of the root surface. REFERENCES BHAT, K. K. S. & NYE, P. H. (1973). Diffusion of phosphate to plant roots in soil. 1. Quantitative autoradiography of the depletion zone. Plant and Soil, 38, 161. BHAT, K. K. S. & NYE, P. H. (1974). Diffusion of phosphate to plant roots in soil. III. Depletion around onion roots without root hairs. Plant and Soil, 41, 383. BREWSTER, J. L., GANCHEVA, A. N. & NYE, P. H. (1975). The determination of desorption isotherms phosphate using low volumes of solution and an anion for soil exchange resin. Journat of Soil Science, 26, 364. GERDEMANN, J. W. (1968). Vesicular-arbuscular mycorrhiza and plant growth. Annual Review of Phytopathology, 6, 397. GERDEMANN, J. W. (1975). VA mycorrhizae. In: Development and Ftmction of Roots (Ed, by J. G. Torrey & D. T. Clarkson), pp. 575-591. Academic Press, London. GERDEMANN, J. W. & NICHOLSON, T. H. (1963). Spores of mycorrhizal Endogone species extracted from soil by wet sieving and decanting. Transactions of the British mycological Society, 46, 235. GERDEMANN, J. W. & TRAPPE, J. M. (1974). The Endogonaceae in the Pacific Northwest. Mycologia Memoir, 5, pp. 76. HATTINGH, M. J. (1975). Uptake of ^-P-labelled phosphate by endomycorrhizal roots in soil chamber?. In: Endomycorrhizas (Ed. by F. E. Sanders, Barbara Mosse & P. B. Tinker), pp. 289-295. Academic Press, London. HATTINGH, M. J., GRAY, L. E. & GERDEMANN, J. W. (1973). Uptake and translocation of ^^P-labelled phosphate to onion roots by endomycorrhizal fungi. Soil Science, 116, 383. HAYMAN D. S. & MOSSE, B. (1971). Plant growth responses to vesicular-arbuscular mycorrhiza. 1. Growth of Endogone-inoculated plants in phosphate-deficient soils. New Phytologist, 70, 19. JACKSON, M. L. (1966). Soil Chemical Analysis - Advanced Course. Published by author, University of Wisconsin, Madison. JARVIS, R. A. (1968). Soils of the Reading District. Memoirs of the soil survey of England and Wales, Sheet No. 268. LEWIS, D. G. & QUIRK, J. P. (1967). Phosphate diffusion in soil and uptake by plants. III. ^^P-movement and uptake by plants as indicated by ^^P-autoradiography. Plant and Soil, 26, 445. MOSSE, B. (1959). Observations on the extra-matrical mycelium of a vesicular-arbuscular endophyte. Tranactions of the British mycological Society, 42, 439. MOSSE, B. (1973). Advances in the study of vesicular-arbuscular mycorrhiza. Annual Reviezo of Phytopathology, 11, 171. MOSSE, B. & BOWEN, G. D. (1968). The distribution of Endogone spores in some Australian and New Zealand soils and in an experimental soil at Rothamsted. Transactions of the British mycological Society, 51, 485. MOSSE, B., HAYMAN, D. S. & IDE, G. H. (1969). Growth responses of plants in unsterilized soil to inoculation with vesicular-arbuscular mycorrhiza. Nature, 224, 1031. NYE, P. H. (1966). The effect of nutrient intensity and buffering power of a soil, and the absorbing power, size and root hairs of a root, on nutrient absorption by diffusion. Plant and Soil, 25, 81. PEARSON, V. & TINIER, P. B. (1975). Measurement of phosphorus fluxes in the external hyphae of endomycorrhizas. In: Efidomycorrhizas (Ed. by F. E. Sanders, Barbara Mosse & P. B. Tinker), pp. 277-287. Academic Press, London, New York. PHILLIPS, J. M. & HAYMAN, D. S. (1970). Improved procedures for clearing roots and staining parasitic and vesicular-arbuscular mycorrhizal fungi for rapid assessment of infection. Trajisactions of the British mycological Society, 55, 158. POWELL, C. LL. & DANIEL, J. (1978). Mycorrhizal fungi stimulate uptake of soluble and insoluble phosphate fertilizer from a phosphate-deficient soil. New Phytologist, 80, 351. RHODES, L. H. & GERDEMANN, J. W. (1975). Phosphate uptake zones of mycorrhizal and non-mycorrhizal onions. Nezo Phytologist, 75, 555. SANDERS, F. E. & TINKER, P. B. (1973). Phosphate flow into mycorrhizal roots. Pesticide Science, 4, 385. TEMPLE-SMITH, M. G. & MENARY, R. C. (1977). Movement of ^^P to roots of cabbage and lettuce grown in two soil types. Communications in Soil Science and Plant Analysis, 8, 67. TINKER, P. B. H. (1975). Effects of vesicular-arbuscular mycorrhizas on higher plants. In: Symbiosis, pp. 325-349. Symposium of the Society for Experimental Biology, no. 29.