THE RESPONSE OF MYCORRHIZAL AND NON- MYCORRHIZAL ROOTED CUTTINGS OF HEATHER (CALLUNA VULGARIS (L.) HULL) TO VARIATIONS IN NUTRIENT AND WATER REGIMES

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1 New Phytol. (1974) 73, THE RESPONSE OF MYCORRHIZAL AND NON MYCORRHIZAL ROOTED CUTTINGS OF HEATHER (CALLUNA VULGARIS (L.) HULL) TO VARIATIONS IN NUTRIENT AND WATER REGIMES BY P. BANNISTER AND W. M. NORTON Department of Biology, University of Stirling, and Department of Botany, University of Glasgow {Received 6 August 1973) SUMMARY Rooted cuttings of heather showed their best drj' matter production in drained regimes with a frequent addition of nutrients. Mycorrhizal plants generally showed a decreased production when compared with equivalent nonmycorrhizal plants, except on drained regimes low in nutrients where the mycorrhizal plants grew better. Concentratio of nitrogen in the shoot and potassium and phosphorus in the root were higher in mycorrhizal than in nonmycorrhizal plants, effects most marked in welldrained regimes with the minimum addition of nutrients. In general, alteratio in nutrient and water regimes had more significant effects on dr}' matter production than did the presence or absence of mycorrhiza. It is concluded that mycorrhizal infection may have a beneficial effect in freely drained, infertile, soils that are characteristic of welldeveloped heathland. INTRODUCTION Studies of the growth of ericaceous plants in relation to soil conditio (Bannister, 1964) cannot differentiate between the respoe of the mycorrhizal component and that of the whole plant. Therefore, any respoe may be either due to a direct effect upon the host plant or to an indirect effect involving the mycorrhiza. The ecological status of ericaceous mycorrhizas is still a controversial matter although it seems unlikely that the early views of an obligate, systemic, infection w ith the capacity to fix atmospheric nitrogen are valid (Harley, 1969). As it is possible to obtain rooted cuttings of plants of heather {Calluna vulgaris (L.) Hull) which are free of mycorrhizal infection (Christoph, 1921), this paper compares the respoe of such cuttings with those with mycorrhizas to variatio in nutrient and moisture regimes. MATERIALS AND METHODS Three nutrient regimes and drained or waterlogged moisture regimes were chosen to approximate the range of conditio under w hich heather may be found in the field. The rooting medium was a synthetic mineral medium, 'Peralite', which has virtually no nutrientsupplying capacity of its own. This was chosen in preference to sterilized soil because of the suggestio that autoclaved soils and other organic media inhibit the growth of ericaceous seedlings and that this inhibition is overcome by the presence of 81

2 82 p. BANNISTER AND W. M. NORTON mycorrhizal or even nonmycorrhizal fungi (Knudson, 1933; Freisleben, 1935; Bain, 1937). Thus any apparent stimulation of growth could be interpreted merely as a release from such inhibition. We also found a 40% inhibition of shoot exteion in rooted cuttings on autoclaved mixtures of peat and sand as compared with identical mixtures which were not autoclaved (unpublished data). Shoots of Calluna vulgaris, collected from Mugdock Moor, near Milngavie, 10 km north of Glasgow (Nat. Grid Ref. NS ) were rooted in sterile sand and found to be free of mycorrhizal infection; the criterion for infection being the presence of hyphal knots in the cortical cells of the finer roots. Some rooted cuttings were infected by growing them, for a period of approximately 2 weeks, with mycorrhizal plants collected from the field. A subsequent development of infection was observed in all cuttings treated in this manner. The mycorrhizal and nonmycorrhizal cuttings were traplanted into 4in plastic plant pots containing 'Peralite' and nutrients added as 10 cm* aliquots of Knop's solution (Curtis and Clark, 1950). Aliquots were added at intervals of i, 3 or 6 weeks to give three nutrient regimes. Waterlogging was obtained by impeding the drainage in some pots. The twelve basic treatments (mycorrhizal and nonmycorrhizal plants on three nutrient and two water regimes) were randomly positioned in each of five blocks in an unheated glasshouse. The experimental period was from December 1969 to November 1970, when height, horizontal spread, number of branches and dry weight of shoots and the dry weight of roots were measured. The majority of these parameters were subjected to logarithmic traformation prior to statistical analysis in order to secure a more symmetrical distribution about their mea. The roots were also examined for mycorrhizal infection. Bulked samples of the five replicates of each treatment were dried at 90 C and then powdered in a hammer mill. Subsamples (normally 400 mg but less in some cases) were digested in 4.4 cm^ of a mixture of sulphuric acid and hydrogen peroxide (6:5 v/v) with lithium sulphate (18.2 mg cm~^) and selenium (0.54 mg cm~^) (K. Taylor, personal communication) and made up to 50 cm^ with deionized water. Lanthanum chloride in a final concentration of 400 ppm was added to the digests prior to analysis with a Unicam SP90 spectrophotometer. Sodium and potassium were measured by emission spectrophotometry and calcium and magnesium by atomic absorption on digests which were normally diluted five times. Undiluted digests (0.5 or i cm'') were used for the colorimetric determination of nitrogen (Williams, 1964) and phosphorus (Allen, 1940). At least two separate determinatio of each element were made from the digests. RESULTS Respoes to nutrient and water regime The main parameters used in the statistical analyses, namely shoot height and shoot, root and total dry weights are all highly intercorrelated and their analyses (Tables i and 2) are discussed jointly. The first two blocks showed a higher total and root dry weight than the rest; this was related to their position in the glasshouse. There was no difference in shoot height and dry weight between blocks. There was a highly significant difference (Table 2) in growth in relation to nutrient regime. The greatest production was in the pots with the weekly application of nutrients but there was less difference between the other two rates of application. Dry matter

3 Mycorrhizas and varying soil conditio 83 production was better in the drained regimes although there was no significant difference in shoot height. There was no evidence for any interaction between nutrient and water regime. The horizontal spread and the number of branches per shoot were both greater in the drained regimes and the pots with highest nutrient application. Table i. Mean values {logarithmic units) of various measures of performance of heather cuttings grown with and without mycorrhizal infection over a range of nutrient and water regimes Mea Shoot height Shoot weight Root weight Total weight Shoot height (log mm) Shoot weight (log mg) Root weight (;iog mg) Total weight (log mg) Shoot height Shoot weight Root weight Total weight Waterlogged Drained Nonmycorrhizal1, drained NonmycorrhizalI, waterlogged Nonmycorrhiza! 1 Mj'corrhizal Ill Mycorrhizal f drained Mycorrhizal waterlogged ni I I I n,, nutrient application every 6 weeks, ni, extry 3 weeks, n3, everv' week. Each measurement is the average of five replicates n Table 2. Results of analyses of variance for various measurements made on heather cuttings grozvn with and without mycorrhizal infection on a range of water and nutrient regimes Source of variation Blocks Infection (I) Water regime (W) Nutrient regime (N) IxW IxN IxWxN Shoot Shoot Root Total Leader Branch Horizontal height weight weight length number spread *** ** * weight ** * **, P<o.ooi; **, P<o.oi; *, P<o.O5;, not significant, P>o.O5. The highest shoot concentratio of sodium (Fig. ia), calcium (Fig. id), and phosphorus (Fig. if) are found in the waterlogged regime, whereas the highest shoot concentration of potassium is in plants from the drained regime (Fig. ib). There is no difference in the tissue concentration of either magnesium (Fig. ic) or nitrogen (Fig. le) with respect to moisture regime. The tissue concentratio of sodium, potassium and calcium in the roots are all highest in the waterlogged regime whilst there are no significant differences in the root concentration of other elements with respect to water regime (Table 3).

4 Drained Woter logged n, rij 03 1 Shoot Root (0) L J (b) r) : : 5 0 (d) _ _ (e) (f) Fig. I. Tissue concentration (mg g ~' dry weight) of various elements in roots and shoots of heather subjected to different nutrient and water regimes, (a) Sodium; (b) potassium, (c) magnesium; (d) calcium; (e) nitrogen; (f) phosporus. rij, nutrient application every 6 weeks; n2, every 3 weeks; n3 weekly. The presence of mycorrhizal infection is indicated by stippling. The significance of differences is indicated in Table 3. "2

5 Mycorrhizas and varying soil conditio ai CO K en ~ U c««c c c o I 1 a C5 iri Vi Q &C.M B B o I ^ C* B C B a C C B B B B B 1 I EA«* m eft en C C B B eg 1^ C* B C * O B 1) *j O.2 ^.2 >H il x^o S 2 o 41 V

6 86 P. BANNISTER AND W. M. NORTON The greatest absolute amount of any element is usually found in the treatments with the highest nutrient application (Table 4) but these treatments may have the lowest tissue concentration. This effect is particularly marked with respect to sodium and calcium concentratio (Fig. ia, d). In contrast, the phosphorus (Fig. if) and, to a lesser extent, the nitrogen concentratio (Fig. ie) increase with increasing nutrient application. There is some interaction between nutrient and water regimes (Table 3). Decreases in sodium concentration with respect to nutrient application are less marked in drained regimes (Fig. ia) whereas the increase of concentration of nitrogen and phosphorus with increased nutrient application is most marked in drained regimes (Fig. ie, f). Table 4. Mineral content of heather plants (Fig. i) expressed on a wholeplant basis (expression as absolute content (/.ig per plant) and (in parentheses) as concentration (mg g " ^ dry weight) Treatment Drained, mycorrhizal Waterlogged, mvcorrhizal Drained, nonmvcorrhizal Waterlogged, nonmycorrhizal n^ Dry wt (mg) III i Na 267 ( ( ( ( ( ( (j ( ( ( ( (133 K Mg Ca ' t; ^ Each value is the mean of five replicates. ni, nutrient application every 6 weeks; n2, every 3 weeks; nj, weekly. N P ) ) ) ) ) ) ) ) ) ) ) ) Effects of mycorrhiza An examination of the roots at the end of the experiment showed that the 'nonmycorrhizal' treatments were free of mycorrhiza and that the mycorrhiza had been retained in the infected series. There was no apparent difference in the degree of mycorrhizal infection with respect to either nutrient or moisture regime. The effects of mycorrhiza on performance were not marked. There was usually some depression of shoot height, shoot dry weight and total dry weight of mycorrhizal plants compared with the equivalent treatment without mycorrhiza. The greatest depression of shoot height was in mycorrhizal plants on waterlogged soils and waterlogged soils were generally associated with the poorest performance of mycorrhizal plants (Table i). However, there is evidence for a beneficial effect of mycorrhiza on the performance of plants on the lowest, drained, nutrient regime. The root, shoot and total dry weight were all higher than in any other low nutrient treatment (Table i).

7 Mycorrhizas and varying soil conditio 87 There are only a few effects of mycorrhiza on tissue nutrient concentratio (Table 3). The sodium concentration in the roots of mycorrhizal plants shows less marked difference with respect to nutrient regime than in nonmycorrhizal plants (Fig. ia) whilst there is an increased root concentration of potassium, particularly in drained regimes (Fig. ib). There is an increased concentration of nitrogen in the shoots and, to a lesser extent, in the roots of mycorrhizal plants; particularly on drained regimes (Fig. ie). Shoot concentratio of phosphorus are decreased, particularly in drained regimes and at higher levels of nutrient application. There is an increased concentration of phosphorus in the roots of mycorrhizal plants at low levels of nutrient application and particularly in drained regimes (Fig. if. Table 3). This effect is lost or reversed at higher levels of nutrient application. Although there are significant effects of the mycorrhiza on the mineral content and general performance of the heather plants in this investigation, the most significant effects are due to alteratio in nutrient and water regime (Tables 2 and 3). DISCUSSION Relatively little work has been done on the nutrient requirements of Calluna (Gimingham, 1972). The increase in growth which occurs when nutrients are added indicates that nutrients were limiting in this experiment and that Calluna can respond favourably to nutrient addition, in contrast to Erica tetralix L. which tolerates only low concentratio of nutrients in solution (Sheikh, 1969). There is a positive correlation (r = , P <o.o5) between the dry weight of plants and their phosphorus concentration, suggesting that phosphorus limited growth in this experiment. No positive correlatio between concentration and growth were found for any other element, although significant negative correlatio were found for sodium and calcium. These are probably related to the greater proportion of woody tissue (with a lower concentration per unit dry weight than in leafy tissue) in the larger plants. Magnesium is unlikely to have a major influence on the growth of Calluna as there were almost no significant differences between experimental treatments. The poorer growth of mycorrhizal plants in wet regimes and at higher levels of nutrient application is comparable with the respoe of plants with ectotrophic mycorrhizas (Harley, 1969). Some authors (Stahl, 1900; Christoph, 1921; Singh, 1964) have reported little difference in the growth of mycorrhizal and nonmycorrhizal ericaceous plants while Morrison (1957) usually found better growth in nonmycorrhizal plants of Pernettya macrostigma Col., a result similar to our own. These findings involved reasonably mature plants whereas reports of stimulation of growth in mycorrhizal ericaceous plants are often from work with young seedlings (Rayner, 1927) or from plants grown on highly organic, autoclaved, media (Wolf, 1954). The interpretation of these results must take account of the different respoes of germinating seedlings and established plants and of possible inhibition by organic substances in autoclaved media (Knudson, 1933; Freisleben, 1935; Bain, 1937). However, the experiment reported here does indicate that some stimulation of growth occurs in welldrained mineral media of a low nutrient content, i.e. the conditio for the best development of heathland in the field (Gimingham, 1972). Thus, mycorrhizas may be of advantage to the heather plant in its natural environment. Calluna is only rarely found on very fertile soils in the field, but is frequent in wet conditio. We have found

8 88 P. BANNISTER AND W. M. NORTON less intee mycorrhizal infection in plants from wet sites so that any inhibitory effect may be unimportant. The increased concentration of nitrogen in the shoots and of phosphorus and potassium in the roots of mycorrhizal plants may explain the stimulation of growth which occurs in drained conditio with a low nutrient supply. Nitrogen and phosphorus are in particularly short supply in most heathland soils (Gimingham, 1972). The accumulation of phosphorus in mycorrhizal roots of Calluna is similar to that found by Pearson (1971) using '^P and provides a parallel with the enhanced uptake of phosphorus by ectotrophic mycorrhizas (Harley, 1969). The accumulation of phosphorus in mycorrhizal roots at the expee of shoots (Pearson, 1971) makes it difficult to see how growth can be enhanced unless the phosphorus is made available to the shoot. This could be accomplished by the trafer of phosphorus from the mycorrhizal fungus to the host. Smith (1966) has demotrated such a trafer with an orchid endophyte, whilst Pearson (1971) has found that ^^P moves from root to shoot when a plant is given a further application of nonradioactive phosphorus. Alternatively, phosphorus could be supplied by the digestion of the fungus. This is most rapid during the growing season (Rayner, 1927). Aaron (1965) has shown a traient peak of phosphorus concentration in leafy shoots of Calluna in early spring which is coistent with a sudden increase in availability when digestion first becomes rapid. Potassium also appears to be accumulated in the roots of mycorrhizal plants and we found a maximum concentration of 4.6 mg g~ * potassium in June which contrasted with a January minimum of 2.7 mg g~ ^ These field observatio may be explained in a variety of other ways, but they are coistent with the hypothesis that the digestion of mycorrhizal fungus in spring leads to higher shoot concentratio of potassium and phosphorus. The accumulation of phosphorus in mycorrhizal roots may provide a strategic reserve of phosphorus analogous to that postulated for Australian heath plants (Jeffrey, 1964). The observatio contained in this paper support the opinion that ericaceous mycorrhizas are of advantage in field conditio as the optimal development of heathland occurs on welldrained mineral soils deficient in nitrogen and phosphorus. However, under waterlogging or high nutrient supply, the possession of a heavy mycorrhizal infection is of dubious advantage and may even be deleterious. ACKNOWLEDGMENTS We should like to thank Professor P. W. Brian and Professor J. H. Burnett who gave advice and encouragement during their terms of office at Glasgow University. The work was carried out during the tenure of an S.R.C. award which provided financial support for apparatus and a research assistantship (W.M.N.). One of us (P.B.) would like to thank Professor F. G. T. Holliday for the provision of facilities which allowed the continuation of the project at the University of Stirling. REFERENCES AARON, J. R. (1965). Studies on the mineral nutrient status of heather, Calluna vulgaris. Forest Rec, Lond., 53, I ALLEN, R. T. L, (1940). The estimation of phosphorus. Biochem. J., 342, 358. BAIN, H. F. (1937). Production of synthetic mycorrhiza in the cultivated cranberry.^, agric. Res., 55, 811. BANNISTER, P. (1964). The water relatio of certain heath plants with reference to their ecological amplitude. I. Introduction: germination and establishment. X EcoL, 52, 423. CHRISTOPH, H. (1921). Untersuchungen (iber die mycotropben Verhaltnisse der 'Ericales' und die Keimung von Pyrolaceen. Beih. bot. ZbL, 38, 115.

9 Mycorrhizas and varying soil conditio 89 CURTIS, O. F. & CLARK, D. G. (1950). An Introduction to Plant Physiology. New York, McGrawHill. FREISLEBEN, R. (1935). Weitere Untersuchungen iiber die Mykotrophie der Ericaceen. J'6. wiss. Bot., 80, 421. GIMINGHAM, C. H. (1972). Ecology of Heathlands. Chapman and Hall, London. HARLEY, J. L. (1969). The Biology of Mycorrhiza, 2nd edn. Leonard Hill, London. JEFFREY, D. W. (1964). The function of polyphosphate in Banksia ornata, an Australian heath plant. Aust. J. biol. Sci., 17, 845. KNUDSON, L. (1933). N'onsymbiotic development of seedlings of Calluna vulgaris. New Phytol., 32, 115. MORRISON, T. M. (1957). Hostendophyte relatiohip in mycorrhiza of Pernettya macrostigma. New Phytol., 56, 247. PEARSON, V. (1971). The biology of the mycorrhiza in Ericaceae. Ph.D. thesis. University of Sheffield. RAYNER, M. C. (1927). Mycorrhiza. New Phytol., reprint no. 15. SHEIKH, K. H. (1969). The effects of competition and nutrition on the interrelatio of some wetheath plants, y. EcoL, 57, 87. SINGH, K. G. (1964). Fungi associated with the roots and rhizosphere of Ericaceae. Ph.D. thesis. University of Durham. SMITH, S. E. (1966). Physiology and ecology of Orchis mycorrhizal fungi with reference to seedling nutrition. New PAy^o/., 65, 488. STAHL, E. (1900). Der Sinn der Mykorrhizenbildung. J'6. wiss. Bot., 85, 151. WILLIAMS, P. C. (1964). The colorimetric determination of total nitrogen in feeding stuffs. Analyst, Lond., 89, 276. WOLF, E. (1954). Beitrag zur Systematik der Gattung Mortierella und MortierellaArten als Mykorrhizapilze bei Ericaceen. Zentbl. Bakt. ParasiteKde, 107, 523.

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