A. K. Sharma, P. C. Srivastava* and B. N. Johri**^,†

Directorate of Extension, *Department of Soil Science, College of Agriculture and **Department of Microbiology, C.B.S.H., G.B. Pant University of Agriculture and Technology, Pantnagar 263 145, India

Uptake and translocation of phosphorus (³²P) by mycorrhizal (Glomus macrocarpum) and non-mycorrhizal Sesbania aculeata L. was studied in a short-term solution culture experiment at low (2 ´  10^–4 k mole P m^–3) and high (1 ´  10^–3 k mole P m^–3) P concentrations. Uptake in mycorrhizal Sesbania was more sensitive to 2,4 dinitrophenol, especially under low P supply. Total P uptake in plants at different time intervals conformed to a Langmuir type equation. The calculated maximum P uptake capacity of the mycorrhizal plants was 1.83 and 1.61-fold higher than the non-mycorrhizal plants under low and high P supply, respectively. During the early duration (0.25 h), P accumulation by root and its translocation to stem was greater and more metabolically-dependent in mycorrhizal than non-mycorrhizal plants. This effect was more evident under low P supply. At a later phase (8 h), P accumulation in roots of mycorrhizal plants remained highly metabolic but the translocation to stem and leaves became much less dependent on cell metabolism.

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VESICULAR-arbuscular mycorrhizae (VAM) are reported to improve P nutrition of many leguminous crops¹. In soil culture, mycorrhizal roots absorb P from labile pool by extending extramatricular hyphae beyond the zone of P depletion in the vicinity of roots^2–4. The actual rate of phosphorus removal from the soil solution is largely determined by the amount of the absorptive surface area¹, a factor which is quite variable among plant species. Root absorption capacity, the absorption rate per unit root measured under standardized conditions of concentration, temperature, etc. may play some role in determining the rate of phosphorus uptake, particularly if phosphate is very abundant⁵. On most occasions, however, the phosphate concentration of the soil solution is so low and diffusion so slow that root absorption kinetic properties play a negligible role^5,6. In a solution culture study, Cress et al.⁷ demonstrated that VAM were capable of absorbing P more efficiently by greater affinity for phosphate carrier systems than those of root alone. Phosphate absorption by VAM has been shown to be temperature sensitive⁸. However, most such investi-

†For correspondence. (e-mail: bnj_bbm@gbpuat.ernet.in)

gations have involved root tissues only. The present study reports the effect of VAM fungi on time dependent P uptake kinetics of Sesbania aculeata L. plants under both low and high P supply. The effect of 2,4 dinitrophenol (DNP), a metabolic inhibitor on P accumulation by different plant parts was also examined.

The inoculum of Glomus macrocarpum was multiplied on maize for three months. Spores counts were made after wet sieving and decanting method⁹.

A riverbed coarse sand (8 kg) was made nutrient-free by soaking it in a mixture of 10% HCl and 1% oxalic acid for 7 days followed by repeated washing with deionized water until it was acid free based on pH change. Acid-washed sand was sterilized by autoclaving for 2 h

at 121° C and filled in eight plastic pots of 500 g capacity. Spores of G. macrocarpum were surface sterilized employing 0.1% streptomycin, 0.1% gentamycin, 0.2% chloramine T and 0.1% tween 80 according to Tawaraya et al.⁹. Four pots were inoculated by placing 200 spores in each, 1 cm below the root tips of pre-germinated seeds of S. aculeata; other half of the pots served as control. Pots were transferred to a glasshouse with day and night temperature maxima of 35° C and 20° C. Plants were raised on distilled water for first 10 days and at half-strength modified Hoagland solution containing half the P level thereafter. After 20 days of growth, young plants were gently removed after flooding the pot with deionized water and washed to remove adhering sand parti-

cles. Seedlings of uniform vigour were selected for P uptake study. Fifty randomly selected plant roots from inoculated and control plants were stained with acid fuchsin¹⁰ and examined under a microscope (´  100) for mycorrhizal infection by the method of Biermann and Lindermann¹¹.

Half-strength modified Hoagland solution (free of P) was prepared in glass distilled water. Five hundred millileters of this solution containing 2 ´  10^–4 k mole P m^–3 (low P) or 1 ´  10^–3 k mole P m^–3 (high P) with or without 1 ´  10^–2 k mole DNP were taken in conical flasks in triplicate. To each flask, carrier-free radioactive (³²P) was added at the rate of 18.17 m Ci/flask. Five plants wrapped in a cotton plug were fitted to each flask in such a manner that the roots remained dipped in the radioactive solution. The flasks were covered with black paper and kept in a growth chamber at 25 ±  1° C under artificial light (730 m mol m^–2 s^–1); contents were aerated by bubbling compressed air to each flask.

Plants were removed from the flasks after 0.25, 0.5, 1, 2, 3, 4, 6 and 8 h. Roots were washed in demineralized water for 15 min followed by another 5 min wash in half-strength modified Hoagland solution containing cold P level corresponding to that of the treatment. Plants were blotted between the filter paper sheets, separated into leaves, stem and roots and dried at 70° C for 48 h in an electric oven and dry weights were recorded. Plant parts were digested in tri-acid mixture (HNO₃:H₂SO₄:HClO₄, 10:1:4 V/V) and diluted to 25 ml in volumetric flask. An aliquot of 0.5 ml was used to determine the activity of ³²P on a gas proportional counter.

Roots of inoculated S. aculeata showed 67.8% infection whereas control roots were free of any fungal infection.

Phosphorus uptake in plants increased with change in the treatment duration at both low and high P supply (Figure 1). The initial uptake rate of mycorrhizal plants was greater than the non-mycorrhizal plants but pres-

 

ence of DNP decreased it at both, low and high P supply levels. Bowen et al.⁸ have reported the active nature of P absorption by mycorrhizal fungi.

Under low P supply conditions, the extent of inhibition of P uptake was higher in mycorrhizal plants than non-mycorrhizal control. However, at the high P supply, the extent of DNP inhibition of P uptake appeared more or less similar for both mycorrhizal and non-mycorrhizal plants. Since P uptake values followed more or less a hyperbolic pattern with respect to uptake period, P uptake data were fitted to the linear form of a Langmuir type equation Q_t^–1 = Q_max^–1 + B/Q_max t^–1, where Q_t = P uptake (n moles g^–1 dry weight) by plants at time t (h), Q_max = maximum P uptake capacity and, B = a constant corresponding to time (in h) required to achieve the half of the maximum P uptake.

While fitting the data of non-mycorrhizal plants in the absence of DNP and under high P supply in this equation, P uptake values at 0.5 and 2 h were considered outliners and rejected. Phosphorus uptake by both mycorrhizal and non-mycorrhizal plants fitted well in the above equation (r = 0.72 to 0.98, all significant at P = 0.05) (Table 1). Under low P supply, the maximum P uptake capacity (Q_max) of mycorrhizal plants was 1.83 fold higher than the non-mycorrhizal plants and the former required only half the time of the latter to attain the half of the maximum P uptake capacity. Under high P supply, a similar trend was noted for Q_max and B values but the differences were less spectacular. Based on calculated values of Q_max and B, P influx (n mole P g^–1 tissue s^–1) was calculated as Q_max/2B ´  3600. In the absence of DNP, the computed P influx varied in the range of 0.31 to 6.92 n moles g^–1 s^–1. These P influx values tally closely to the earlier reported values (0.1–3.8 n mole cm^–2 s^–1), measured either directly in sterile dual cultures^3,12 or those derived theoretically in pot culture experiments¹³. The calculated P influx of mycorrhizal plants was 260 and 65% higher than the non-mycorrhizal plants under low and high P supply, respectively. Under solution culture, where P transfer from growing medium to the root surface is not limited, increased P uptake rate of mycorrhizal plants could be ascribed to their higher P uptake capacity⁷. The presence of DNP decreased P influx and the extent of inhibition was greater in mycorrhizal plants compared to non-mycorrhizal plants. This signifies that P influx in mycorrhizal plants is driven by metabolically-dependent active processes. The transport of P to the host roots by vesicular-arbuscular fungi has been shown to be driven by metabolically-dependent active processes involving protoplasmic streaming and to a lesser extent by bulk flow through hyphal content¹².

Under low P supply, P concentration build up in root and stem of mycorrhizal plants at an early stage (0.25 h) was greater and more sensitive to DNP inhibition compared to that of non-mycorrhizal plants (Figure 2 a). At high P supply too, during the early stages mycorrhizal plants accumulated higher concentration of P in root and stem than the non-mycorrhizal plants; however, the

 

Figure 2 b.  Effect of VAM on P uptake in root, stem and leaves of Sesbania aculeata under P deficient and sufficient conditions.

effect of mycorrhization and DNP inhibition was less pronounced. This signifies that under low P supply mycorrhizal roots absorbed and translocated higher amount of P by metabolically-dependent mechanism. At a later stage (8 h), P concentration in roots of mycorrhizal plants under both low and high P supply was greater and more sensitive to DNP inhibition than non-mycorrhizal plants (Figure 2 b). Phosphorus concentration in stem and leaves of mycorrhizal plants under both low and high P supply was also less sensitive to DNP inhibition than non-mycorrhizal plants. This may be ascribed to higher acquisition of P by mycorrhizal roots and its subsequent bulk flow under water potential gradient of the transpiring shoots. Recently, Smith et al.⁶ have concluded that an enhanced efflux from fungus to the host plant was essential for symbiotic uptake of P by plants via VA mycorrhizal fungi.

Thus, mycorrhizal S. aculeata plants, under low P supply absorb more P than non-mycorrhizal plants due to a metabolically-dependent higher P influx. A Langmuir type kinetic equation could adequately describe time dependent uptake kinetic of P in the plant species. Phosphorus accumulation in mycorrhizal roots occurs through a metabolically-dependent process, however, translocation of absorbed P to stem and leaves is much less dependent on this component.

 

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ACKNOWLEDGEMENTS.  We thank the Incharge, Radio Isotopic Tracer Lab. of the university, for providing necessary facilities to carry out this work. We thank Dr K.V.B.R. Tilak of IARI, New Delhi for supplying cultures of Glomus macrocarpum.

 Received 1 July 1999; accepted 21 September 1999