EFFECT OF ENDOGONE MYCORRHIZA ON PLANT GROWTH

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1 New Phytol. (1969) 68, EFFECT OF ENDOGONE MYCORRHIZA ON PLANT GROWTH II. INFLUENCE OF SOLUBLE PHOSPHATE ON ENDOPHYTE AND HOST IN MAIZE BY M. J. DAFT AND T. H. NICOLSON Department of Biological Sciences, University of Dundee {Received 18 April 1969) SUMMARY Varying amounts of soluble phosphate were applied over different periods during the growth of mycorrhizal maize. The amount of the endophyte produced within the root system and the growth of the host were related inversely. Repeated small doses of soluble phosphate over long periods depressed the production of the fungus more than when given over shorter periods. Large single applications of soluble phosphate applied early in the growth of the host also reduced the amount of the endophyte more than when given later. The role of mycorrhiza in relation to phosphate nutrition is discussed. INTRODUCTION The absorption and uptake of inorganic nutrients can be greatly influenced by the rhizosphere microflora and this is particularly so in the case of phosphate (Bowen and Rovira, 1966; Rovira and Bowen, 1966) an ion for which microorganisms in general 'have a great avidity' (Barber, 1968). Mycorrhizas, which can be considered as a special case of rhizosphere effect (Harley, 1948), also influence nutrient uptake especially that of phosphate (Bowen and Theodorou, 1967). The phycomycetous or vesicular-arbuscular mycorrhizas are the most common (Gerdemann, 1968; Nicolson, 1967) and in certain environments phosphate uptake would appear to be dependent on them and hence of great ecological importance (Baylis, 1967; Redhead, 1968). Earlier work showed that when readily available phosphate was supplied in the nutrient solution to plants growing in sand culture, it was difficult to detect differences in growth between mycorrhizal and non-mycorrhizal treatments. However, when the sole phosphate source was slowly available and supplied in low concentrations growth differences were conspicuous. Bone meal was a source which acted in this way as also were apatite, and tricalcium phosphate (Daft and Nicolson, 1966). Similar results have heen noted by Murdoch, Jacobs and Gerdemann (1967) who found that with poorly available sources, such as rock phosphate or tricalcium phosphate, mycorrhizal grew better than non-mycorrhizal plants. On the other hand, with highly-available monocalcium phosphate or super-phosphate they could not detect such differences. The aim of this work was to study the effects of soluble phosphate on the degree of infection and on the growth of the host in mycorrhizal maize. 945

946 M. J. DAFT AND T. H. NICOLSON MATERIALS AND METHODS The host was a single cross maize hybrid and the endophyte was Endogone macrocarpa var. geospora (Nicolson and Gerdemann, 1968).

2 946 M. J. DAFT AND T. H. NICOLSON MATERIALS AND METHODS The host was a single cross maize hybrid and the endophyte was Endogone macrocarpa var. geospora (Nicolson and Gerdemann, 1968). This variety of Endogone was formerly designated spore type 3 (Gerdemann and Nicolson, 1963). It forms conspicuous ectocarpic chlamydospores which are easy to count with a binocular stereoscopic microscope. Germination of the seeds, inoculation and transplanting were performed as reported previously (Daft and Nicolson, 1966). Each pot was supplied twice weekly with 50 ml Long Ashton nutrient solution at half the recommended concentration but with the phosphate salt (KH2PO4) omitted. With treatments receiving soluble phosphate a solution of KH2PO4 was added to the nutrient solution. At harvest the aerial parts of the plants were dried at 100 C and weighed. The endophyte was assessed by recording the number of spores occurring on each individual plant. The roots were washed until all spores were detached and retained on sieves of mesh size 250jU and 150 ju. The two fractions were freed from most of the plant debris by decanting and the spore sample made up to a suitable volume in water. Aliquots were removed from a continuously stirred sample, placed on a filter paper and the spores counted. From this count the total number of spores per replicate was calculated. In one experiment, root infection was also measured by removing i g fresh roots from Table i. Dry weight and mean numbers of spores for mycorrhizal maize plants grown with four different sources of phosphate {plants grown for 10 weeks) Phosphate source Solubility* Mean dry Mean spore weight of no./plant foliage (g) Tricalcium phosphate Sodium metaphosphate Insoluble Magnesium pyrophosphate Insoluble Manganese orthophosphate * Solubilities ex Handbook of Chemistry and Physics. each plant. These samples were then pooled for each treatment and used to prepare 'root slides' for the estimation of percentage infection (Nicolson, i960). Since certain treatments entailed omission of the soluble phosphate salt (KH2PO4) from the nutrient solution, it was necessary to provide a basic 'insoluble' phosphate to give measurable plant growth. Bonemeal, apatite and tricalcium phosphate are suitable sources (Daft and Nicolson, 1966) but the first two compounds can vary in composition. A preliminary experiment tested four chemically defined phosphate sources which were thought to be possible compounds for the basic 'insoluble' phosphate. These were applied at a standard level of 1.2 mg equivalents (m-equiv.) of phosphate per plant. This experiment was done under poor autumnal light conditions in the glasshouse. The results, shown in Table i, indicate that of the four salts only tricalcium phosphate was suitable and it was therefore used throughout this investigation at 1.2 m-equiv./pot. It allowed measurable growth with good mycorrhizal development. The solubilities of the four phosphate sources and the grov^th were not related but there was an inverse relationship between growth and number of spores detached from the roots.

3 Soluble phosphate and Endogone mycorrhiza RESULTS 947 Effect of continuous application of soluble phosphate over varying periods on endophyte development Experiment i. In two experiments soluble phosphate was included in the twice weekly application of the nutrient solution. In the first experiment the seven periods during which it was applied are shown in Fig. i(a). The treatments were as follows. (i) A: control with no soluble phosphate given. (ii) C, D and E: soluble phosphate applied for 2^, 5 and 7^ weeks respectively after inoculation. (a) Plants - inoculated I No I soluble I phosphate Soluble phosphate applied (b) ^ 70 ^\ 60 ^ ^ 30 Is D E P Tl'eatment Fig. I. Effects of soluble phosphate applied over varying periods on the numbers of spores of the endophyte, root infection and dry weight of foliage of mycorrhizal maize, (a) Periods over which soluble phosphate was applied; (b) mean numbers of spores fopen columns) and mean percentage infection (stippled columns); (c) mean dry weights of foliage. (iii) P: soluble phosphate applied for the entire growth period of 10 weeks, (iv) Q, R and S: soluble phosphate applied after 2^, 5 and ']\ weeks respectively had elapsed from the time of inoculation. Because of the large number of replicates involved it was not possible to run equivalent non-mycorrhizal control treatments. The results for this experiment are given in Fig. i (b and c). In general, with longer

4 948 M. J. DAFT AND T. H. NICOLSON periods of soluble phosphate application there was a diminution in production of spores and in percentage infection. This was particularly well defined for percentage infection but less so for numbers of spores. The high mean spore numbers for treatments C and D were due to a few large plants which gave exceptionally high spore counts. Percentage infection on the other hand, was not a mean figure but was estimated from a composite sample and hence such variability between replicates within a treatment was not shown. Plants Or inoculated ~ I 5-6 E i- No soluble phosphate Soluble phosphole applied 10 (b) (c) C D Treatment Fig. 2. Effects of soluble phosphate applied over varying periods on the numbers of spores of the endophyte and dry weight of foliage of mycorrhizal maize, (a) Periods over which soluble phosphate was applied; (b) mean numbers of spores; (c) mean dry weights of foliage. Statistical analysis of spore numbers showed that treatment P (soluble phosphate applied throughout) produced fewer spores than all the other treatments and E {jl weeks soluble phosphate application) produced fewer than A, C, D and S (P<o.o5). The growth of the host plants reflected the length of time soluble phosphate was applied (Fig. ic). Treatments A and S produced smaller plants than E, P and Q and treatments C and R were smaller than Q (P<o.o5). Experiment 2. Since in experiment i the numbers of spores in treatments C and D appeared inconsistent and not related to percentage infection figures another experiment was set up. This consisted of treatments A, C, D, E and P as before (no phosphate

5 Soluble phosphate and Endogone mycorrhiza 949 and 2^, 5, 7 and 10 weeks respectively with phosphate) and an additional treatment where phosphate was applied for i week from inoculation (treatment B, Fig. 2a). Spore numbers decreased gradually with increasing duration of phosphate application (Fig. 2b). The amount of variation amongst the plants was less than that found in experiment i. Host growth again showed increases with increasing phosphate application except that treatment B (i week) was slightly smaller than treatment A (no phosphate). This may show that the greater mycorrhizal development in A more than compensated for the small amount of phosphate given in treatment B. Both experiments show, in (a) Plants -0 inoculated (b) (c) 2 i; 3 Z 5- I r S 4000 a. (/) 3000 i g 2000 s 1000 p I I 0o S 7 E 6 Ul u 0 3 '' U09IAI T 00 C n) < n >r t. Y Z 1 1 n No soluble phosphate Soluble phosphate opplied Treatment Fig. 3. Effects of a single application of soluble phosphate at various times on the nunribers of spores of the endophyte and dry weight of foliage of mycorrhizal maize, (a) Times at which single applications of soluble phosphate were made (<-); (b) mean number of spores; (c) mean dry weight of foliage in mycorrhizal (open columns) and control (stippled columns) plants. general, an inverse relationship between plant size and mycorrhizal development as in the preliminary experiment (Table i). Effects of a single application of soluble phosphate on endophyte development In another experiment soluble phosphate was applied to each treatment on one occasion only. The amount of soluble phosphate (1.2 m-equiv.) given at the only application was equivalent to that used throughout treatment P (Fig. ia). Treatments W, X, Y and Z received the single soluble phosphate application i, 2, 5 and 7 weeks respectively after inoculation (Fig. 3a).

950 M. J. DAFT AND T. H. NICOLSON The results are given in Fig. 3 (b and c). Fewer spores were produced when the total phosphate was applied after 2 weeks from inoculation than after i week.

6 950 M. J. DAFT AND T. H. NICOLSON The results are given in Fig. 3 (b and c). Fewer spores were produced when the total phosphate was applied after 2 weeks from inoculation than after i week. Further delay in the application of phosphate resulted in an increase in the number of spores. Again, the maximum number of spores was produced in the treatment that received no phosphate (A). Dry weight decreased as the application of phosphate was delayed (Fig. 3c). In this experiment non-mycorrhizal control plants were included in each phosphate treatment but there was no size difference between infected and non-infected plants in any of them (Fig. 3c). Only in the no phosphate treatment (A) was there a significant difference between mycorrhizal plants, with a mean dry weight of 3.8 g and control plants with a mean of2.2g(p<o.oi). This shows that the stimulus to the host conferred by the mycorrhiza is overcome by the addition of phosphate. DISCUSSION The experiments reported here further indicate the close relationships which exist between vesicular-arbuscular mycorrhiza and phosphate nutrition. In the case of an annual host it was possible by differential timing of soluble phosphate applications to manipulate the amount of mycorrhizal infection. Endophyte activity, as estimated both by numbers of spores and percentage infection, was progressively depressed when soluble phosphate was applied over increasing periods. The same was true when the maximum quantity of phosphate was given early during growth of the host. These results clearly show the effects of soluble phosphate on the amount of infection produced after 10 weeks growth ofthe host. The effects of the infection on the uptake of phosphate is possibly shown only in one of the experiments. In experiment 2 (Fig. 2c), treatment A (no phosphate) produced slightly larger plants than treatment B (phosphate for I week, 0.12 m-equiv.) and treatment A also produced more spores (Fig. 2b). Treatment C (phosphate for 2^ weeks, 0.30 m-equiv.) produced plants larger than treatment A. It would seem possible then, that the benefit of the highest infection resulted in a greater rate of phosphate uptake and this benefit was equivalent to the effects of an amount of phosphate between 0.12 and 0.30 m-equiv. No cumulative effect of high mycorrhizal infection and the addition of soluble phosphate could be distinguished. Even the addition of soluble phosphate after 7^ weeks (treatment Z, Fig. 3c) in a total period of 10 weeks, nullified the beneficial effects of infection since there was no statistical difference in the dry weights between the mycorrhizal and control plants. However, in spite of the striking relationship between phosphate and the vesiculararbuscular association shown here it should be stressed, as has been done for mycorrhizas in general (Bowen and Theodorou, 1967), that they should not be regarded solely as being involved with phosphate uptake. Several workers have reported increased absorption for a number of other mineral nutrients (Baylis, 1959; Gerdemann, 1964; Mosse, 1957)- There was general agreement between spore numbers and percentage infection (Fig. ib). The size of the plants was inversely related to these estimates of the endophyte (Figs, ib and c, 2b and c). A single apphcation of phosphate had a more marked effect on the endophyte than the recurring applications. The difference between the least number of spores (treatment X) and the maximum (treatment A) was six-fold with the former method of application (Fig. 3), whereas it was only three-fold (treatments P and A) with the latter (Fig. i). In the case of the single applications, phosphate after 2 weeks (treatment X) depressed the endophyte more than with the i week application (treat-

Soluble phosphate and Endogone mycorrhiza 951 ment W, Fig. 3). The reason for this is obscure. It may be related to the stage reached in the infection process.

7 Soluble phosphate and Endogone mycorrhiza 951 ment W, Fig. 3). The reason for this is obscure. It may be related to the stage reached in the infection process. Phosphate given at this time could alter the environmental conditions within the rhizosphere or the physiology of the host roots, in such a way as to be unfavourable to the establishment of the endophyte. Since vesicular arbuscular mycorrhizas benefit host growth more under low phosphate conditions and this benefit is not apparent with the application of soluble phosphate, it may not be of importance in agriculture when good fertility levels are maintained. However, with phosphate fertilizers only some 20 30% is recovered in the first crop following the year of application with the remainder then becoming fixed.* This residual phosphate evidently continues to be utilized by plants even up to years thereafter and this may be beneficial in preserving phosphate for later crops (Cooke, 1965; Sauchelli, 1965). The reason for this continued availability* has not been satisfactorily explained. It may be that mycorrhiza is an important factor in the utilization of such 'fixed' phosphate. This is a possibility which is rarely, if ever, considered in fertilizer or pedological investigations. Rorison (1968), using solution culture techniques, has shown that ecologically distinct species varied in their response to soluble phosphate. From this work it seems probable that each plant species has its own particular phosphate requirement for optimum growth. Hence, in studying the influence of phosphate in the ecology of a species, it is necessary to consider its inherent absorptive capacity together with the possible enhanced uptake when it is mycorrhizal. The degree of infection itself will be influenced by the phosphate fertility of the soil. As stressed previously (Nicolson, 1967), it is in natural habitats that vesicular-arbuscular mycorrhiza is likely to be of greatest significance. Baylis (1967) has indicated the immense ecological importance of these mycorrhizas in woody hosts of the New Zealand rain forest. Indeed, he considers that the association is an adaptation for obtaining 'difficultly available phosphate' and further states 'phycomycetous endophytes assist in the uptake of phosphate from soils far below the minimum standard of fertility'. Maintenance of fertility in natural communities requires either the addition of elements to replace those lost by leaching or an efficient means of recycling. Nitrogen is an element which is lost by leaching but can be replaced by both asymbiotic and symbiotic fixation from the atmosphere. With phosphorus there is a need for recycling as there are no natural processes by which it can be increased substantially after depletion. Mycorrhiza may be the means by which an efficient economy is maintained. Phosphorus from plant remains is quickly converted into an unavailable form in soils and hence is less likely to be lost by leaching. In this form it may then be available to the next generation of plants only if they are mycorrhizal. Hence, in a number of natural environments phosphorus may be a limiting factor in plant growth and it may be that mycorrhiza is the essential link in the cycling of this key metabolic element. As nodulation has been evolved as a mechanism for nitrogen fixation, vesicular-arbuscular mycorrhiza may have been evolved as a means for the more efficient extraction of phosphorus from the pedosphere. ACKNOWLEDGMENTS We are grateful to Professor J. L. Harley, F.R.S. for his helpful criticism of the manu- * The terms 'fixation' and 'availability' are widely used with reference to soil phosphate (Anon., 1965; Jackson, 1962). However, their usage has been criticized by Larsen (1967) who states '... both words have in the past been interpreted in so many ways that they no longer have a generally accepted meaning.' It is difficult to find suitable alternatives.

8 952 M. J. DAFT AND T. H. NICOLSON script and to Mrs L. Blair and Miss E. Davidson for technical assistance. We are also grateful to the Science Research Council for financial assistance. REFERENCES ANON. (1965). Soil Phosphorus. Technical Bulletin No. 13. London. BARBER, D. A. (1968). Micro-organisms and inorganic nutrition of higher plants. A. Rev. Pt. PhysioL, 19, 71. BAYLIS, G. T. S. (1959). Effect of vesicular-arbuscular mycorrhizas on growth of Griselinia littoralis (Cornaceae). New Phytol., 58, 274. BAYLIS, G. T. S. (1967). Experiments on the ecological significance of phycomycetous mycorrhizas. New Phytol, 66, 231. BowEN, G. D. & RoviRA, A. D. (1966). Mfcrobial factor in short term phosphate uptake studies with plant roots. Nature, Lond., 211, 665. BowEN, G. D. & THEODOROU, C. (1967). Studies on phosphate uptake by mycorrhizas. XIV. lupro. Kongress, Mtichen., 116. COOKE, G. W. (1965). The responses of crops to phosphate fertilizers in relation to soluble phosphorus in soils. In: Soil Phosphorus, p. 64. Technical Bulletin No. 13. London. DAFT, M. J. & NICOLSON, T. H. (1966). Effect of Endogone mycorrhiza on plant growth. New PhytoL, 65, 343. GERDEMANN, J. W. (1964). The effects of mycorrhiza on the growth of maize. Mycotogia, 56, 342. GERDEMANN, J. W. (1968). Vesicular-arbuscular mycorrhiza and plant growth. A. Rev. Pt. Path., 6, 397. GERDEMANN, J. W. & NICOLSON, T. H. (1963). Spores of mycorrhizal Endogone species extracted from soil by wet sieving and decanting. Trans. Br. mycot. Soc, 46, 235. HARLEY, J. L. (1948). Mycorrhiza and soil ecology. Biot. Rev., 33, 127. JACKSON, M. L. (1962). Soit Chemical Analysis. London. LARSEN, S. (1967). Soil phosphorus. Adv. Agron., 19, 151. MossE, B. (1957). Growth and chemical composition of mycorrhizal and non-mycorrhizal apples. Nature, Lond., 179, 922. MURDOCH, C. L., JACOBS, J. A. & GERDEMANN, J. W. (1967). Utilization of phosphorus sources of different availability by mycorrhizal and non-mycorrhizal maize. PL Soil, 27, 329. NICOLSON, T. H. (i960). Mycorrhiza in the Gramineae. II. Development in different habitats, particularly sand dunes. Tram. Br. mycol. Soc, 43, 132. NICOLSON, T. H. (1967). Vesicular-arbuscular mycorrhiza a universal plant symbiosis. Sci. Prog., Oxf., SS, S6i. NICOLSON, T. H. & GERDEMANN, J. W. (1968). Mycorrhizal Endogone species. Mycologia, 60, 313. REDHEAD, J. F. (1968). Mycorrhizal associations in some Nigerian forest trees. Trans. Br. mycol. Soc, RORISON, I. H. (1968). The response to phosphorus of some ecologically distinct plant species. I. Growth rates and phosphorus absorption. New Phytol., 67, 913. RoviRA, A. D. & BowEN, G. D. (1966). Phosphate incorporation by sterile and non-sterile plant roots. Aust. J. biol. Sci., 19, SAUCHELLI, V. (1965). Phosphates in Agriculture. New York.

EFFECT OF ENDOGONE MYCORRHIZA ON PLANT GROWTH

New Phytol. (1969) 68, 953-963. EFFECT OF ENDOGONE MYCORRHIZA ON PLANT GROWTH III. INFLUENCE OE INOCULUM CONCENTRATION ON GROWTH AND INFECTION IN TOMATO BY M. J. DAFT AND T. H. NICOLSON Department of Biological