PHOSPHATASE ACTIVITY ASSOCIATED WITH THE ROOTS AND THE RHIZOSPHERE OF PLANTS INFECTED WITH VESICULAR-ARBUSCULAR MYCORRHIZAL FUNGI

Transcription

1 New Phytol. (1987) 17, ' \',. \. ^ ;^::;;;;T*^ - y3--^;- ^ ^ '\ : -^ ""'^ 163 PHOSPHATASE ACTIVITY ASSOCIATED WITH THE ROOTS AND THE RHIZOSPHERE OF PLAS INFECTED WITH VESICULAR-ARBUSCULAR MYCORRHIZAL FUNGI BY J. C. DODD*, C. C. BURTON, R. G. BURNS AND P. JEFFRIESf Biological Laboratory, University of Kent, Canterbury, Kent CT2 INJ, UK {Accepted 1 May 1987) SUMMARY Acid phosphatase activity associated with the root and in the rhizosphere of rape {Brassica napus) L., wheat {Triticum aestivum L.) and onion {Allium cepa L.) plants was investigated in the presence or absence of vesicular-arbuscular mycorrhizal fungi. The plants were inoculated singly with three different species of Glomus, or were left uninfected, and were grown in sand with little available phosphorus. Root and rhizosphere levels of phosphatase activity were higher for plants infected with (Nicol. & Gerd.) Gerd. & Trappe and G. mosseae (Nicol & Gerd.) Gerd. & Trappe compared with control plants. Infection with Gerd. & Trappe did not result in a similar increase in phosphatase activity but significant increases in plant growth nevertheless occurred. Further experiments, using wheat only, showed that increases in phosphatase activity induced by infection by or G. mosseae became apparent between 25 and 51 d after sowing. Key words: Vesicular-arbuscular mycorrhizas, soil enzymes, phosphatase, rhizosphere, Glomus. IRODUCTION The beneficial growth responses conferred on mycotrophic host plants by vesicular-arbuscular mycorrhizal (VAM) fungi have been explained primarily in terms of improved uptake of phosphate (Nye & Tinker, 1977; Mosse, Stribley & LeTacon, 1981; Hayman, 1983). The chief attribute of VAM fungi is their ability to absorb soluble phosphate (and other nutrients) beyond the phosphatedepletion zone that develops around the root surface. This zone is particularly marked and arises in soils of low phosphorus (P) status because of the poor mobility of phosphate ions in the soil (Tinker, 198; Hayman, 1983). The external hyphae absorb nutrient ions and translocate them to the root (Pearson & Tinker, 1975). Phosphate is taken up and probably translocated as polyphosphate granules by protoplasmic streaming (Cox et al., 1975) before being hydrolyzed in the arbuscules prior to transmembrane transfer into the host cell (Capaccio & Callow, 1982). Phosphatase enzymes are also directly involved in the acquisition of phosphorus by plants. Their importance, however, is not always obvious. The proposition that plants with lower activities of root phosphatase may gain and use phosphorus more readily than plants with higher ones has been put forward by McLachlan (1976, 198a, b), who found that acid phosphatase activity was lower in plants * Present address: Soil Microbiology Department, Rothamsted Experimental Station, Harpenden, Herts., U.K. t Author to whom correspondence should be sent X/87/ $3./ 1987 The New Phytologist

2 164 J. C.DoDD et al. more efficient at P uptake when grown under P-deficient conditions. Mycorrhizal infection has been shown to influence phosphatase activity. Increased uptake of P by ectomycorrhizal trees, for example, has been attributed to the increased root surface area but, in addition, phosphatase activity has been implicated (Bartlett & Lewis, 1973 ; Alexander & Hardy, 1981; Antibus etal., 1981). Acid phosphatases produced by the ectomycorrhizal fungal mantle and the attached ramifying mycelium are believed to catalyze the hydrolysis of complex organic phosphorus compounds into more readily absorbed forms (Ho & Zak, 1979). Ridge & Rovira (1971) indicated that root surface phosphatases may be more important in the organic P mineralization than enzymes in the aqueous phase or bound to the surrounding soil. Antibus et al. (1981) showed that the acid phosphatase activity of ectomycorrhizal roots of Salix rotundifolia Trautv. colonized by Entoloma sericeum (Bull, ex Merat) Quelet. was 4 times as great as non-mycorrhizal roots on a surface area basis. Dighton (1983), on the other hand, showed a decrease in phosphatase activity produced by mycorrhizal birch roots compared with nonmycorrhizal controls, but also demonstrated high extracellular phosphatase production by several ectomycorrhizal fungi, suggesting their potential for degradation of complex phosphorus-containing compounds. Acid phosphatases have also been reported in VAM fungi and although their function is unclear they may be associated with the growth and development of the fungus within the host tissue (Gianinazzi, Gianinazzi-Pearson & Dexheimer, 1979) as well as with phosphorus aquisition in the rhizosphere. MacDonald & Lewis (1978), for example, detected acid phosphatase activity in VAM fungal structures both inside and outside roots using a cytochemical method. Azcdn, Borje & Barea (1982) compared the effects of VAM infection and phytate addition on the growth, P uptake and surface acid phosphatase activity of wheat and lavender roots (species differing in mycorrhizal dependency under low P conditions). Mycorrhizal infection decreased the activity of root surface phosphatase of lavender compared with non-mycorrhizal control plants, but it had no effect on wheat. Similarly, Gianinazzi-Pearson & Gianinazzi (1986) reported that VAM infection of clover did not significantly affect the activity of root surface acid phosphatase, but they did note that phytase activity was increased and that this effect varied with the fungus involved. We report here an investigation of acid phosphatase activity associated with the root and in the rhizosphere of VAM-infected and uninfected plants in sand culture in an attempt to discover if phosphatase activity is related to mycorrhizal dependency under low nutrient conditions. The experiment was also designed to determine if the ' efficiency' of a particular VAM isolate is related to its ability to increase rhizosphere levels of phosphatases. Three plants were used to cover the range of mycotrophic responses, namely rape [a non-mycorrhizal species which has been shown to be efficient in absorbing soil P (Bhat, Nye & Baldwin, 1976)], wheat [low mycorrizal dependency in soils of low P status (Mosse, 1986)] and onion [high mycorrhizal dependency in soils of low P status (Mosse, 1986)]. ' M A T E R I A L S AND M E T H O D S Inoculation and growth of plants Rape {Brassiea napus L. var. Hungry Gap), onion {Allium cepa L. var. Ailsa Craig) and wheat (Triticum aestivum L. var. Avalon) seeds were germinated on glass fibre paper (Whatman GF/A) moistened with sterile, distilled water. After

3 Phosphatases in VA mycorrhizal plants d, four germinated seeds with similar radicle development (i.e. 5 to 1 mm) were planted 2 cm below the surface of the growth medium. The plants were grown in a constant temperature room (2 C, 16 h day/8 h night, 6 % RH). The plants were irrigated with one-third strength Hoagland's nutrient solution without orthophosphate (Hoagland & Arnon, 1939) In experiment 1, germinated seeds of rape, onion and winter wheat were sown in 15 mm diameter polyethylene pots containing 1-75 kg autoclaved builders' sand [ph 8 and 7 mgl"^ NaHCOg-soluble P (Olsen et al, 1954)]. Inoculum, from maize (Zea mays L.)-grown pot cultures of three VAM fungal isolates: Glomusgeosporum (Nichol. & Gerd.) Walker (isolate number: UKC M l ), Glomus mosseae (Nicol. & Gerd.) Gerd. & Trappe (UKC M2) and Glomus monosporum Gerd. & Trappe (UKC M3) was throughly mixed into the sand at a rate of 15 g dry weight per pot. This amount of inoculum was considered large enough to override differences in relative infectivity between species. Inoculum consisted of spores and fragmented, infected root material. s were inoculated with equivalent amounts of autoclaved inoculum of each isolate which had been soaked in a filtrate from inoculum of all three endophytes which was free from infective VAM propagules. Six replicate pots were used for each treatment. Plants were thinned after 1 week to three per pot for rape, two per pot for onion and four per pot for wheat. Harvests were at 53, 72 or 98 d for wheat, rape and onion, respectively. In the second experiment, germinated seeds of winter wheat (Avalon) were sown in an autoclaved, coarse builders' sand (ph 7 5). Inoculum (3 g) of each of the three VAM fungi was mixed into each pot as above. Six replicates were again used for each treatment. Plants were thinned to four per pot after 7 d. Harvests were made at 21, 51 and 74 d. Rhizosphere and root sampling procedure The plants were carefully removed from the pots and the root systems plus adhering sand were excised from the shoots. The roots were then shaken by hand until most of the sand was detached (outer rhizosphere sample) and then washed with sterile, distilled water (1 to 1 ml depending on the weight of the root-sand mass) to remove the small amount of strongly adhering sand (inner rhizosphere sample). The remaining roots were excised before being assayed for phosphatase activity. Sand without plants, but otherwise treated similarly to that in which plants were grown, served as the non-rhizosphere control. Samples were taken with a 1 cm diameter soil borer at the same time as rhizosphere samples. Assessment of plant growth and VAM infection After harvesting, shoot and root fresh weights were determined. In addition, shoot dry weights were recorded in both experiments (dried at 8 C for 24 h). For assessment of infection, fresh roots were washed, laid flat and a 1 cm segment half way along each root excised. Root segments were cleared and stained with trypan blue (Phillips & Hayman, 197) using lactoglycerin instead of lactophenol and percentage VAM infection was assessed by the grid-line intersect method (Giovanetti & Mosse, 198) using a low-power ( x 5) binocular microscope. Figures quoted are means of five replicate plants. Assay of phosphatase activity Acid phosphatase activity (E.C orthophosphoric-monoester phos-

4 ^.... ;. :,, M J. C. D O D D e^ a/.:.^^;k%^.:''#'''. phohydrolase) was measured using a modification of the method of Tabatabai & Bremner (1969). Samples (1 g dry weight) of the inner and outer rhizosphere sand and the control sand were incubated with 1 ml 5 mm ^-nitrophenyl phosphate (PNP) and 4 ml 1 M sodium acetate buffer, ph 5 2, for 1 h at 25 C in a shaking water bath (6 rpm). The reaction was terminated by the addition of 5 ml 5 M NaOH and the samples were centifuged at 25 g for 1 min. The optical density of the supernatant was measured at 4 nm in a spectrophotometer (LKB ultraspec 2). Phosphatase activity was expressed as the amount of pnitrophenol released during incubation. s was assayed by adding 1 ml PNP to a suspension containing sand and acetate buffer after 6 min and immediately before the addition of NaOH. This measures endogenous soil components (especially humates) which absorb at 4 nm and any /)-nitrophenol released during NaOH and centrifugation treatment. The standards were prepared in acetate buffer solution containing, 1, 2, 3, 4 or 5 mm /)-nitrophenol. Phosphatase activity associated with intact root segments was measured by determining the amount of/)-nitrophenol released when 1 ml of 5 mm PNP and 4 ml of -1 M sodium acetate buffer, ph 5-2, was incubated with 1 mg of fresh root tissue. Activity recorded represents mainly root surface phosphatase activity with a small component due to diffusion of internal root phosphatase from the cortex and the cut ends of excised roots. The /)-nitrophenol product was developed with NaOH and determined spectrophotometrically in the same way as for rhizosphere phosphatase activity. As far as possible, lengths of root with yellow pigmentation were chosen for determination of phosphatase activity. Such pigmentation is characteristic of dense VAM infection sites (e.g. Becker & Gerdemann, 1977), and this was confirmed by microscopic observations of the root segments for the presence of external mycelium or spores. Statistical analysis of the root and rhizosphere sand enzyme activities were made using the Newman-Keuls multiple range test. The ph activity curves of rhizosphere and root phosphatase were determined after 74 d using a series of buffers of the following composition and p H : 3*8 to 5-8 sodium acetate; 5-7 to 6-8 sodium maleate; 6-5 to 8- tris/maleate; 7-5 to 9-5 tris HCl (all 1 M). All assays were replicated six times. For characterization of the root surface acid phosphatase of plants infected with G. mosseae or uninfected (control), washed roots were assayed after incubation intervals of 3, 6, 9, 12, 18, 21 and 24 min and straight line plots were obtained. The kinetic values for acid phosphatase were derived from Lineweaver-Burk transformations of the Michaelis-Menten equation using activities measured over a range of substrate concentrations (167 to 2 mm). Lines of fit were calculated by least square regression analysis. Correlation coefficients were > -93 in all cases. R E S U L T S AND D I S C U S S I O N Figures 1 (a) and (b) shows ph activity curves of phosphatase activity associated with wheat roots infected by G. mosseae [Fig. 1 (a)] and in the total rhizosphere [Fig. l(b)]. Figure l(a) shows that the root surface has a broad range of activity between ph 5 and 7, with a peak at ph 5 5 to 6. In the rhizosphere, however, there is a more clearly defined peak at ph 5- to 5-5 with reduced activity above ph 6. This indicates that acid phosphatase activity predominates in the rhizosphere and on the roots and that there is very little alkaline phosphatase. A

5 Phosphatases in VA mycorrhizal plants u D <U l/> O O Q. V) o i. 1. O f g I I I I I I 1 ) X (/) _ B I T3 I Q. O I/) Fig. 1. Effect of ph on phosphatase activities either on roots (a) or in rhizosphere (b) of wheat infected with Glomus mosseae. ph was determined using a range of buffers: sodium acetate (A); sodium maleate (A); tris-maleate (O); and tris-hcl (#). similar pattern was observed for plants infected with the other two VAM fungi tested. The results obtained from intact roots correlate with those of Gianinazzi et al. (1979) who used histochemical studies to localize phosphatase activity within VAM-infected roots at an ultrastructural level. Acid phosphatase (ph < 6) activity was observed in the cytoplasm of the plant root cells as well as at the growing tips of mycorrhizal hyphae. Alkaline phosphatase (ph < 8) activity, however, was weak in root cells and was unaflfected by the presence of VAM fungus. McLachlan (198a) found that the optimum ph for surface root phosphatase activity lay in the range ph 5 to 6 when he tested uninfected buckwheat, rye, subterranean clover and wheat, but found no evidence of alkaline phosphatase activity. Activities of acid phosphatase of the rhizosphere and root surface in experiment 1 are shown in Table 1 for each host. Results for onion are from the 98 d harvest, those from rape are from the 72 d harvest, whilst those of wheat are from the 53 d harvest. At these times, adequate plant growth had occurred and there was no significant difference between replicates at any of the harvests in terms of shoot dry weights or enzyme activity. As expected, rape did not become infected with VAM fungi. Onion was more difficult to analyze than the other two plants due to its slow rate of growth and paucity of root material produced, and therefore the rhizosphere was not divided into inner and outer zones (even at 98 d).

6 168 J. C. DoDD et al. Table 1. Acid phosphatase activities {\xmol p-nitrophenol 1 mg root tissue^ \imol p-nitrophenol 1 mg root tissue'^ h~^ or \imol p-nitrophenol g sandt^ h after 53 (wheat), 72 (rape) or 98 (onion) d Plant Treatment VAM Infection (%) Dry weight Shoot (g) per pot Root surface Enzyme activity Inner Outer rhizosphere rhizosphere Rape LSD, P = 5 Wheat LSD, P = -5 Glotnus mosseae Glomus mosseae NS * * * * 1-7* * * * -3 Onion LSD, P = -5 Glomus mosseae * -8* -1* * 4-99* Total rhizosphere * -6* -49-5, not tested for LSD as the difference in percentage infection between isolates is not necessarily related to phosphatase activity; NS"^, F value not significant; *, significant difference from control (P = -5). Activities of phosphatase in the rhizosphere were higher for plants infected with G. mosseae and (i.e. wheat and onion) compared with the control plants. Infection by, however, did not increase enzyme activity associated with the root or in the inner rhizosphere of any of the plants, but nevertheless a significant (three-fold) increase in shoot dry weight was noted in onion compared with the poorly infected controls. Wheat plants infected with G. mosseae grew better than the controls and had significantly higher activities of root surface phosphatase. plants had become contaminated to a low percentage infection by extraneous or residual VAM infection by 53 d, so no further measurements were made. The comparatively high activities of phosphatases associated with the root and in the rhizosphere of non-mycorrhizal rape compared with either the mycorrhizal onions or mycorrhizal wheat should also be noted. If the inner and outer rhizosphere fractions of rape and of wheat are combined and averaged, they can be compared with each other and with onion. Thus, phosphatase activity in the rhizosphere of rape averages 8-5 x greater than that of wheat and 1-5 x greater than that of onion. The corresponding comparisons for root surface phosphatase vs rhizosphere phosphatases are 24 x and 2-2 X. In experiment 2, the response of wheat to VAM infection was examined in more detail with respect to the age of the plant. Yields, phosphorus concentration in shoot material and the effect on phosphatase activities associated with the root and rhizosphere were monitored. Table 2 shows that the acid phosphatase activity had increased over that of the control in both inner and outer rhizosphere zones and on roots for plants infected with either or G. mosseae after 51 and

7 Phosphatases in VA mycorrhizal plants 169 Table 2. Growth responses and acid phosphatase activities (\imol p-nitrophenol loomg root tissue~^ h~^ or [imol p-nitrophenol g sand~^ h~^) for three successive harvests of winter wheat plants Treatment VAM V xtivl Infection (%) dry weight (g) per pot P (dry weight basis) rt>nr'f*t^1"t*ji1'ion in shoot (/ \ \ /o) Root surface Enzyme activity Inner rhizosphere Outer rhizosphere 25 Days G. mosseae LSD, P = NS+ ND * 2-81* L94* Ml Days G. mosseae LSD, P = * 38-53* * -22* -2* * 617* * 1-34* * -72* Days G. mosseae LSD, P = * -54* -55* * 15* 14* * 7-46* * 2-2* * ND, not determined;, not tested for LSD (see Table 1); NS"^, F value not significant; *, significant difference from control. 74 d (but not by day 25). Significantly, higher internal concentrations of shoot P at days 51 and 74 were noted following infection. In contrast, plants infected with showed no diiferences in root or rhizosphere phosphatase activity from that of the control plants of a similar age, but infection signficantly increased shoot dry weights and almost doubled the phosphorus concentration within the plant shoots. In both these experiments, infection of host plants by certain VAM fungi has been shown to influence activities of root surface and rhizosphere phosphatases. In the second experiment, for example, infection of wheat by two out of the three VAM isolates tested resulted in a significant increase in phosphatase activity in the rhizosphere and on the root when compared with the controls, coinciding with increased growth and higher internal phosphorus concentrations. This effect differs from that noted by Azcon et al. (1982) for VAM-infected lavender in which no increases in activity were noted. Our results using, however, confirm that the isolate of fungus used can be important in determining the phosphatase response. Rape, however, is non-mycorrhizal and the phosphatase activity in or around rape roots should not be affected by the presence of VAM fungi. It is interesting in this respect that in our experiments rape produced the highest levels of extracellular phosphatases when compared with onion and wheat (Table 1). It may be that phosphatase production is inversely related to mycorrhizal

8 I7O A ii J. C. DoBD et al. dependency of a crop species, since root phosphatase activity and the VAM symbiosis could be regarded as having developed through evolution as alternative mechanisms for P uptake by plants growing in low soil P conditions (see also Azcon et at., 1982). Hedley, Nye & White (1983) showed that rape grown in a soil of low P status acidified its rhizosphere, which led to dissolution of an acid-soluble form of inorganic P (Pi) which was not exchangable with the ^^P used in their isotope exchange technique for estimating P taken up from the labile pool. There appears to be a simple process of diffusion of phosphatase enzymes from the root surface to give high activity in the inner rhizosphere of the sand medium. Diffusion increased with time and significant increases in activity are seen in the outer rhizosphere of older plants. It would appear, therefore, that the increased levels of phosphatases noted in the rhizosphere is not a result of direct secretion from external hyphae but occurs due to diffusion from the mycorrhizal root surface. This does not, however, explain the anomaly of, which may increase the rate of P uptake by more efficient uptake or translocation of phosphate, or may have a particularly efficient phosphate transfer system involving less overall production of internal phosphatase activity. Cooper & Tinker (1978) suggested that translocation, rather than uptake of P, is the rate-limiting step in total transfer of P into the host plant and it is possible that can translocate P at a faster rate than either of the other two isolates and, as a result, an increase in phosphatase levels in the rhizosphere does not occur. Alternatively, it may be hypothesized that if could ' tap' sources of Pi unavailable to the non-mycorrhizal plants or even those infected with or G. mosseae (and therefore not deplete the Pi in the labile pool) the plant would not be severely P-deficient and an increase in phosphatase activity would not take place. The and G. mosseae symbioses may show increased phosphatase activity at the root surface as a direct result of depleting the available Pi supply. Our experiments were carried out in coarse sands with low concentrations of bicarbonate-soluble P and the source of the increased shoot P concentrations observed in experiment 2 in VAM-infected plants is unclear and deserves further study, since such observations have been made before (Dodd, Krikun & Haas, 1983). Models for phosphorus uptake based on in vitro measurements of physicochemical parameters controlling phosphate diffusion in soil apparently work well for plants supplied with sufficient phosphorus, but underestimate P uptake by phosphorus deficient plants grown in soils in which concentrations of soluble phosphorus are sensitive to ph fiuctuations (Hedley et al., 1983). The isolate used in these experiments has consistently been the most efficient of the three isolates in stimulating wheat growth and P uptake in greenhouse experiments (Dodd, 1986) and was originally isolated from a low phosphorus soil, whereas the other two endophytes were isolated from a soil with a higher nutrient regime. Our results also suggest that rhizosphere phosphatase activity is unrelated to the VAM external network and is a result of increased root surface phosphatase activity and some limited diffusion of activity into the rhizosphere. Obviously in soil the diffusion and survival of enzymes is likely to be minimal due to adsorption, denaturation and proteolysis (Burns, 1983). Furthermore, our kinetic studies (Table 3) show that not only do the absolute levels of acid phosphatase change with time but that their kinetic properties are altered. The increase in K^^ and ^max following infection by G. mosseae means that the affinity of the enzyme(s) for the substrate has decreased, although the rate of substrate catalysis has increased.

9 Phosphatases in VA mycorrhizal plants 171 Table 3. K^ and ^max "^^lues of acid phosphatase{s) on the roots and in the rhizosphere of 74 d old wheat plants infected with Glomus mosseae or uninfected {control) Roots Rhizosphere G. mosseae * mm. ^ fimol p-nitrophenol released (1 mg fresh weight of root)"^. ^ fitciox /)-nitrophenol released (g sand)"^ h~'. Correlation coefficients > -93 in all cases. This suggests either that the amount of the enzyme(s) has changed (Burns, 1982) and/or that the enzyme(s) that make up the phosphatase activity are different. The role of phosphatases in the soil would obviously depend on the concentrations and chemical state of soil phosphorus as well as the chemical structure of the soil itself. Alexander & Hardy (1981) showed that the activity of surface phosphatases of Sitka spruce ectomycorrhizas was inversely proportional to available concentration of organic phosphorus but trees still grew poorly due to an apparent deficiency of inorganic P. They suggested that, in their investigations, high phosphatase activity could not compensate for an adequate supply of labile inorganic P. The experiments described here have been carried out in autoclaved growth media and irrigated with a sterile nutrient solution. It is likely, nevertheless, that a limited root microflora would have developed as the experiment progressed and that some of the phosphatase activity may have been associated with these microorganisms. Besides having a direct effect on root phosphatases, it is reasonable to consider that VAM infection could cause changes not only in rhizosphere populations, but also in the activity of the microflora at the root surface and in the rhizoplane. The presence of a more diverse rhizosphere microflora in field-grown plants adds another dimension to this picture and this complex relationship awaits investigation. REFERENCES ALEXANDER, I. J. & HARDY, K. (1981). Surface phosphatase activity of Sitka spruce mycorrhizas from a serpentine site. Soil Biology and Biochemistry, 13, AIBUS, R. K., CROXDALE, J. G., MILLER, O. K. & LINKINS, A. E. (1981). Ectomycorrhizal fungi of Salix rotundifolia. III. Resynthesized mycorrhizal complexes and their surface phosphatase activities. Canadian Journal of Botany, 59, AzcoN, R., BoRiE, F. & BAREA, J. M. (1982). Exocellular acid phosphatase activity of lavender and wheat roots as affected by phytase and mycorrhizal inoculation. In: Les Mycorhizes: Biologie et Utilization. Les Colloques de I'INRA, vol. 13 (Ed. by S. Gianinazzi, V. Gianinazzi-Pearson & A. Trouvelot), pp INRA, Paris. BARTLETT, E. M. & LEWIS, D. H. (1973). Surface phosphatase activity of mycorrhizal roots of beech. Soil Biology and Biochemistry, 5, BECKER, W. H. & GERDEMANN, J. W. (1977). Colorimetric quantifications of vesicular-arbuscular infection in onion. New Phytologist, 78, BHAT, K. K. S., NYE, P. H. & BALDWIN, J. P. (1976). Diffusion of phosphate for plant roots in soil. IV. The concentration distance profile in the rhizosphere of roots with root hairs in a low P soil. Plant and Soil, 44, BURNS, R. G. (1982) Enzyme activity in soil: location and a possible role in microbial ecology. Soil Biology and Biochemistry, 14, 423^27.

10 172 ]. C.BoDD et al. (1983). Extracellular enzyme-substrate interactions in soil. Symposium of the Society for General Microbiology, 34, CAPACCIO, L. C. M. & CALLOW, J. A. (1982). The enzymes of polyphosphate metabolism in vesiculararbuscular mycorrhizas. New Phytologist, 91, COOPER, K. M. & TINKER, P. B. (1978). Translocation and transfer of nutrients in vesicular-arbuscular mycorrhizas. II. Uptake and translocation of phosphorus, zinc and sulphur. New Phytologist, Cox, G. C, SANDERS, F. E., TINKER, P. B. & WILD, J. (1975). Ultrastructural evidence relating to hostendophyte transfer in a vesicular-arbuscular mycorrhiza. In: Endomycorrhizas (Ed. by F. E. Sanders, B. Mosse & P. B. Tinker), pp Academic Press, New York & London. DIGHTON, J. (1983). Phosphatase production by mycorrhizal fungi. Plant and Soil, 71, DODD, J. C. (1986). V-A mycorrhizas of winter cereals in S.E. England: ecology, taxonomy and effects of agrochemicals. Ph.d. thesis. University of Kent, UK. DODD, J. C, KRIKUN, J. & HAAS, J. (1983). Relative effectiveness of indigenous populations of vesiculararbuscular mycorrhizal fungi from from sites in the Negev. Israeli Journal of Botany, 32, GIANINAZZI, S., GIANINAZZI-PEARSON, V. & DEXHEIMER, J. (1979). Enzymatic studies on the metabolism of vesicular-arbuscular mycorrhiza. III. Ultrastructural localisation of acid and alkaline phosphatases in onion roots infected by Glomus mosseae (Nicol. & Gerd.) Gerd. & Trappe. New Phytologist GIANINAZZI-PEARSON, V. & GIANINAZZI, S. (1986). The physiology of improved phosphorus nutrition in mycorrhizal plants. In: Physiological and Genetical Aspects of Mycorrhizae. First European Symposium on Mycorrhizae (Ed. by V. Gianinazzi-Pearson & S. Gianinazzi), pp INRA, Paris. GiovANETTi, M. & MOSSE, B. (198). An evaluation of techniques for measuring vesicular-arbuscular mycorrhizal infection in roots. New Phytologist, 84, HAYMAN, D. S. (1983). The physiology of vesicular-arbuscular endomycorrhizal symbiosis. Canadian Journal of Botany, 61, HEDLEY, M. J., NYE, P. H. & WHITE, R. E. (1983). Plant induced changes in the rhizosphere of rape {Brassiea napus var. Emerald) seedlings. IV. The effect of rhizosphere phosphorus status on the ph, phosphatase activity and depletion of soil phosphorus fractions in the rhizosphere and on the cationanion balance in the plants. New Phytologist, 95, Ho, I. & ZAK, B. (1979). Acid phosphatase activity of six ectomycorrhizal fungi. Canadian Journal of Botany, 57, HoAGLAND, D. R. & ARNON, D. I. (1939). The Water-Culture Method for Growing Plants Without Soil. California Agricultural Experiment Station Circular No MACDONALD, R. M. & LEWIS, M. (1978). The occurrence of some acid phosphatases and dehydrogenases in the vesicular-arbuscular mycorrhizal fungus Glomus mosseae. New Phytologist, 8, MCLACHLAN, K. D. (1976). Comparative phosphorus responses in plants to a range of available phosphorus situations. Australian Journal of Agriculture Research, 27, MCLACHLAN, K. D. (198a). Acid phosphatase activity of intact roots and phosphorus nutrition in plants. I. Assay conditions and phosphatase activity. Australian Journal of Agricultural Research, 31, 429^4. MCLACHLAN, K. E. (198b). Acid phosphatase activity of intact roots and phosphorus nutrition in plants. II. Variations among wheat roots. Australian Journal of Agricultural Research, 31, MOSSE, B. (1986). Mycorrhiza in sustainable agriculture. Biological Agriculture and Horticulture, MOSSE, B., STRIBLEY, D. P. & LETACON, F. (1981). Ecology of mycorrhizae and mycorrhizal fungi. Advances in Microbial Ecology, 5, NYE, P. H. & TINKER, P. B. (1977). Solute movement in the Soil-Root System. Blackwell Scientific Publications, Oxford. OsLEN, S. R., COLE, C. V., WATANABE, F. S. & DEAN, L. A. C. (1954). Estimation of Available Phosphorus in Soils by Extraction with Sodium Bicarbonate. US Department of Agriculture Circular No PEARSON, V. & TINKER, P. B. (1975). Measurement of phosphorus fluxes in the external hyphae of endomycorrhizas. In: Endomycorrhizas (Ed. by F. E. Sanders, B. Mosse & P. B. Tinker), pp Academic Press, New York & London. PHILLIPS, J. M. & HAYMAN, D. S. (197). Improved procedures for clearing roots and staining parasitic and vesicular-arbuscular mycorrhizal fungi for rapid assessment of infection. Transactions of the British Mycological Society, 55, RIDGE, E. H. & ROVIRA, A. D. (1971). Phosphatase activity of intact young wheat roots under sterile and non-sterile conditions. New Phytologist, 7, TABATABAI, M. A. & BREMNER, J. M. (1969). Use of/)-nitrophenol phosphate for assay of soil phosphatase activity. Soil Biology and Biochemistry, 1, TINKER, P. B. (198). Role of rhizosphere micro-organisms in phosphorus uptake by plants. In: The Role of Phosphorus in Agriculture (Ed. by F. E. Kwasahneh, E. C. Sample & E. J. Kamprath), pp American Society of Agronomy, Madison, Wisconsin. BURNS, R. G.

11