THE EFFECT OF TEMPERATURE ON THE TRANSLOCATION OF PHOSPHORUS BY RHIZOPUS STOLONIFER

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1 New Phytol. (1969) 68, THE EFFECT OF TEMPERATURE ON THE TRANSLOCATION OF PHOSPHORUS BY RHIZOPUS STOLONIFER BY A. J. E. LYON* AND R. L. LUCAS Department of Agriculture, Oxford {Received 22 April 1969) SUMMARY The effect of temperature on the rate of phosphorus translocation, on the uptake and metaholism of phosphate and on respiratory rate of Rhizopus stolonifer, has heen studied. Over the temperature range C the rate of phosphorus translocation is greatest at 20 C and lowest at 30 C. The rate of oxygen uptake is greatest at 25 C. The rate of phosphate uptake does not vary significantly over this temperature range, hut the proportion of ahsorhed phosphate present as orthophosphate bears a direct relationship to the rate of phosphorus translocation. These results are consistent with the view that phosphorus may he translocated in the form of orthophosphate. INTRODUCTION Many fungi translocate a variety of nutrients through established vegetative mycelium (Thrower and Thrower, 1968; Wilcoxson and Sudia, 1968). Radioisotopic techniques have been used to demonstrate the effect of environmental factors on the rate of translocation in several species (Littlefield, Wilcoxson and Sudia, 1965; Monson and Sudia, 1963; Subbarayudu, Wilcoxson and Sudia, 1966; Wilcoxson and Subbarayudu, 1968). Such studies have shown that the amount of ^ ^phosphorus translocated per unit time may be influenced by temperature, but have made no distinction between its effect on phosphorus uptake and metabolism, and on phosphorus translocation. It is not possible to draw valid conclusions concerning the translocation process itself from measurements of the amount of ^ ^P moved unless it is known how much isotope is present in the hyphae in a form available for translocation. The experiments described in this paper were designed to examine the effect of temperature on translocation, uptake and metabolism of phosphorus and on respiratory rate in Rhizopus stolonifer (Ehrenb. ex Fries) Lind. METHODS Translocation In order to study translocation of nutrients through established mycelium, a modification of the 'split-plate' technique described by Lucas (i960) was used. Potato dextrose agar (PDA) was poured into a sterile polystyrene petri dish. A channel 5 mm wide was removed aseptically along a diameter of the dish. A spore suspension of * Present address: The Department of Botany, The University, Sheffield Sio 2TN.

2 964 A. J. E. LYON AND R. L. LUCAS Rhizopus stolonifer was inoculated on to one-half of each plate and the plate was then incubated at 25 C in darkness. Within 2 days the colony had formed a mycelial bridge across the channel and had colonized the whole plate. A disc of mycelium and agar 13 mm in diameter was removed from the uninoculated half, leaving a well into which were pipetted 0.2 ml of sterile solution containing o.ooi g KH2PO4 labelled with 2 ^Ci ^^P. Plates were incubated for a further 24 hours at 20, 25 or 30 C. Each plate was then sampled by cutting eight 5 mm square samples of mycelium and agar in a strip along a diameter on the inoculated side at right angles to the channel (see Fig. i). Each sample was transferred to an aluminium planchet, dried under infra-red lamps and the beta activity determined using an end-winder Geiger Miiller tube. The amount of phosphorus derived from the added orthophosphate was expressed in micrograms for each sample and the total from eight samples in each strip determined. Well Sampled strip Channel E Sample no. Fig. I. Culture method and presentation of results. There are two sources of error which tend to make this figure an under-estimate of the amount of phosphorus actually translocated. Eirst it is assumed that there is negligeable dilution of the added ^ ^orthophosphate solution by orthophosphate already present in the medium. Secondly it appears from the work of Harley, Brierley and McCready (1954) and Jennings (1964) that orthophosphate is taken up into a small orthophosphate pool from which it may be either translocated or transferred to a larger orthophosphate pool or converted to organic form. The larger the size of the small orthophosphate pool, the greater will be the dilution of the added ^^P by unlabelled phosphorus and the greater the underestimate of total P translocated. Respiration A spore suspension of R. stolonifer was inoculated into 50 ml conical flasks containing 20 ml of 2% glucose in potato extract. The fiasks were incubated at 25 C in a shaking bath for 24 hours. The colonies were then removed from the culture medium by filtering through Whatman No. 50 paper under suction. The colonies were dried by blotting them gently between sheets of filter paper and the fresh weight was determined.

3 Temperature and phosphorus translocation in Rhizopus 965 Each colony was transferred to a Warburg flask containing 5 ml of i % glucose solution. The Warburg flasks were equilibrated for 2 hours at 20, 25 or 30 C. The taps of the manometers were then closed and readings of O2 uptake taken every 15 minutes for 3 hours. Phosphate uptake R. stolonifer was inoculated at the centre of sterile cellophane discs 78 mm in diameter on plates of PDA. The plates were incubated at 25 C for 2 days, by which time the cellophane discs were covered by mycelium. The cellophane was peeled from the agar and transferred to plates of PDA incorporating g KH2PO4 labelled with 0.05 ^Ci ^^P. These plates were incubated at 20, 25 or 30 C for 24 hours. They were then sampled in the following manner. The cellophane was peeled from the medium and washed in cold distilled water to remove adhering Hj^^PO". Mycelial discs 22 mm in diameter were cut from the margin of each colony, transferred to aluminium planchets and dried under infra-red lamps. The dry weight and radioactivity of each disc were determined. Phosphorus metabolism Mycelia of R. stolonifer on cellophane prepared as above were transferred to plates of PDA incorporating o.ooi g KH2PO4 labelled with 2 ^Ci ^^P. Groups of plates were incubated at 20, 25 or 30 C for 24 hours. The mycelium was then scraped from the cellophane, washed in cold distilled water to remove adhering H2^^P0~ and extracted by the method of Benson (1955). Each colony was ground with 2 ml of boiling 80% ethanol for 5 minutes. The resulting macerate was centrifuged at 4000 revs/minute for 15 minutes. The supernatant was decanted into a McCartney bottle and the residue extracted with i ml of 20% ethanol for 5 minutes. The combined extracts were stored at 10 C to precipitate any solid material which otherwise would be deposited on the starting line during subsequent chromatography. After 2 hours the extracts were removed from the deep-freeze and centrifuged in the McCartney bottles. After reducing their volumes under infra-red lamps, the extracts were analysed by descending paper chromatography on acid-washed Whatman No. i paper using picric acid/tertiary butanol/water (2.2 g/80 ml/20 ml) solvent (Hanes and Isherwood, 1949). Chromatograms were scanned with a geiger detector to locate the position of radioactive compounds (Loughman and Martin, 1957)- RESULTS Translocation The efl^ect of temperature on the total amount of translocated phosphorus crossing the mycelial bridge in 24 hours and subsequently detected in the sampled strip is shown in Table i. Each figure is the mean of ten replicates. Measurements of the mycelial bridge fresh weight at the three temperatures show that the amount of mycelium through which translocation was taking place was the same in the three treatments. Significantly more (P<o.o2) phosphorus was translocated at 20 than at 25, and more (P<o.i) at 25 than at 30 C. The distribution of translocated phosphorus within the sampled strip (Fig. 2) appears to be little affected by temperature, except in sample 8, where there is a greater accumulation of translocated phosphorus at 25 than at 20 or

4 A. J. E. LYON AND R. L. LUCAS 30 C. This accumulation corresponds to the development of sporangia at the margin of the plate (Lyon, 1968). Table i. The effect of temperature on the amount of phosphorus translocated by Rhizopus stolonifer in 24 hours Temperature ( C) Mean total fig translocated P Mean mycelial bridge fresh weight (mg) 0-3 2O ±O Fig. 2. The effect of temperature on the distribution of translocated phosphorus in colonies oirhisopus stotonifer. Symbols:, 20 C;, 25 C; A, 30 C. Table 2. Oxygen uptake by colonies of Rhizopus stolonifer incubated at 20, 25 and 30 C O2 taken up/mg fresh weight Temperature ( C) I hour 2 hours 3 hours s Respiration The effect of temperature on oxygen uptake is shown in Table 2. Each value is the mean of four replicates. The rate of uptake of oxygen is greatest at 25 C and lowest at 20 C. Phosphorus uptake Table 3 shows the amount of phosphorus taken up in 24 hours at 20, 25 and 30 C by mycelia of R. stolonifer. Each figure is the mean of ten replicates. No significant

5 Temperature and phosphorus translocation in Rhizopus 967 difference could be detected between the three treatments, but this may be due to the relatively large sampling errors. Phosphorus metabolism Chromatogram scans of mycelial extracts of R. stolonifer incubated on Hj^^PO" at 20, 25 and 30 C are shown in Fig. 3. The proportion of ^^P in the different extracted compounds varies between the three treatments. The activity present in each compound is proportional to the area under the corresponding peak on the chromatogram scan. The relative areas under the peaks are shown in Table 4. ' Table 3. The ejfect of temperature on the amount of phosphorus taken up by colonies of Rhizopus stolonifer in 24 hours Temperature ( C Mean yug P/mg dry weight of mycelium The proportion of activity present in the nucleotide fraction remains relatively constant at the three temperatures. However the proportion of activity present in the form of H2^^P04 falls with increasing temperature, whilst that in the form of hexose phosphate rises over the same temperature range. Origin Nucleotide phosphate Hexose phosphote Fig. 3. Forms of absorbed ^^phospborus in mycelium of Rhizopus stolonifer incubated on labelled medium at tbree different temperatures. Table 4. The relative areas under the peaks in Fig- 3 Relative areas under peaks Front Temperature (" C): HjPOj Hexose-P Nucleotide-P DISCUSSION It is clear that there is no direct relationship between the amount of phosphorus translocated and the respiratory rate at the three temperatures studied. Nor does there appear to be any correlation between the amount of phosphorus translocated and that taken up

6 968 A. J. E. LYON AND R. L. LUCAS at the three temperatures. However, it cannot be assumed that all phosphorus compounds play an equal part in translocation. Harley and Loughman (1963) concluded that ^ ^P was translocated by the hyphae of the fungal sheath of beech mycorrhizas and transferred to the host in the form of orthophosphate. Robinson (1963) and Lyon (1968) have shown that the ability of fungi to translocate phosphorus is dependent on the presence of a labile pool of orthophosphate in the hyphae. In view of these findings it is significant that there is a direct correlation between the amount of phosphorus translocated and the relative level of orthophosphate in the mycelium at the three temperatures (Table 5). This result is compatible with the view that phosphorus is translocated in the form of orthophosphate. If this is the case, factors affecting the size and rate of turnover of orthophosphate pools in the hyphae will infiuence the amount of phosphorus Table 5. The relationship between rate of phosphorus translocation and amount of absorbed phosphate present as orthophosphate Temperature ( C) //g orthophosphate derived from absorbed phosphate in 24 hours (from Tables 3 and 4) fig P translocated in 24 hours (from Table i) Ratio translocated P/orthophosphate i.io available for translocation. For this reason measurements of total phosphorus uptake and translocation under different conditions will not necessarily provide meaningful information about the translocation process itself. Both qualitative and quantitative measurements of the mobile fraction must be made in order to interpret rates of gross movement correctly. ACKNOWLEDGMENTS This work was carried out while one of us (A.J.E.L.) was receiving a Research Studentship from the Ministry of Agriculture. REFERENCES BENSON, A. A. (1955). Phosphorylated sugars. In: Moiiern Methods of Plant Analysis, Vol. II (Ed. by K. Paech & M. V. Tracey). Berlin, Gottingen, Heidelberg. HANES, C. S. & IsHERWOOD, F. A. (1949). Separation ofthe phosphoric esters on the filter paper chromatogram. Nature, Lond., 164, HARLEY, J. L., BRIERLEY, J. K. & MCCREADY, C. C. (1954). The uptake of phosphate by excised mycorrhizal roots of the beech. V. The examination of possible sources of misinterpretation of the quantities of phosphorus passing into the host. New Phytol., 53, 92. HARLEY, J. L. & LOUGHMAN, B. C. (1963). The uptake of phosphate by excised mycorrhizal roots of the beech. IX. The nature of the phosphate compounds passing into the host. New Phytol., 62, 350. JENNINGS, D. H. (1964). Changes in the size of orthophosphate pools in mycorrhizal roots of beech with reference to absorption of the ion from the external medium. New Phytol., 63, 181. LiTTLEFiELD, L. J., WiLCOXSON, R. D. & SuDiA, T. W. (1965). Translocation of phosphorus-32 in Rhizoctonia solani. Phytopathology, 55, 536. LOUGHMAN, B. C. & MARTIN, R. P. (1957). Methods and equipment for the study of the incorporation of phosphorus by intact barley plants in experiments of short duration. J. exp. Bot., 8, 272. LUCAS, R. L. (i960). Transport of phosphorus by fungal mycelium. Nature, Lond., 188, 763. LYON, A. J. E. (1968). Factors affecting the uptake and translocation of nutrients by certain fungi. Dissertation for D.Phil., Oxford University. MoNSON A. M. & SuDiA T.W. (1963). Translocation in Rhizoctonia solani. Bot. Gaz., 124, 440. ROBINSON R. K. (1963). A study of some aspects of the biology of certain root infecting fungi. Dissertation for D.Phil., Oxford University.

7 Temperature and phosphorus translocation in. Rhizopus 969 SUBBARAYUDU S., WILCOXSON R. D. & SUDIA T. W. (1966). Translocation of phospborus-32 in sclerotia oi Phymatotrichum omniverum. Phytopathology, 56, 903. THROWER, L. B. & THROWER, S. L. (1968). Movement of nutrients in fungi, i. The mycelium. Aust. J. Bot., 16, 71. WILCOXSON, R. D. & SUBBARAYUDU, S. (1968). Translocation to and accumulation of pbospborus-32 in sclerotia of Sclerotium rolfsii. Can. jf. Bot., 46, 85. WILCOXSON, R. D. & SUDIA, T. W. (1968). Translocation in fungi. Bot. Rev., 34, 32.

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