THE INOCULATION OF WHITE CLOVER WITH MYCORRHIZAL FUNGI IN UNSTERILE HILL SOILS

Transcription

1 Nev! Phytol. (1982) 92, THE INOCULATION OF WHITE CLOVER WITH MYCORRHIZAL FUNGI IN UNSTERILE HILL SOILS BY A. RANGELEY, M. J. DAFT* AND P. NEWBOULD Hill Farming Research Organisation, Bush Estate, Penicuik, Midlothian, U.K. (Accepted 3 March 1982) SUMMARY The response of two cultivars of white clover (Aberystwyth SI 84 and New Zealand Grasslands Huia), grown under laboratory and field conditions, to inoculation with two endophytes (Glomus mosseae LI and Glomus etunicatus) was investigated in two hill soils with various amounts of added phosphate. In the experiment with the deep peat soil in the laboratory, inoculation with either endophyte increased infection from 2 % to up to 7 %, the increase being less the higher the amount of added P. Shoot growth of white clover was significantly increased (P < -5) by inoculation at the and 2 kg P ha"' level and decreased at the highest level of added P (2 kg P ha~'). Both endophytes produced similar effects. With the brown earth soil, total infection of roots with indigenous endophytes ranged from 3 to 5 % and this was significantly (P < -5) increased by inoculation only at 4 kg P ha"'. However, the introduced coarse endophytes had replaced between a quarter and a half of the predominantly fine indigenous endophyte at each level of added P. This change of endophyte was without significant effect on dry wt of shoots and roots although there was a general trend for inoculation, and with Glomus etunicatus more than with Glomus mosseae LI, to increase shoot and root dry wt. Inoculation also tended to increase the P content of shoots and roots but this was statistically significant only at 4 kg P ha~'. In the field experiment with the deep peat soil, inoculation with Glomus mosseae LI doubled the amount of infected root from 18 to 42 % but there was no effect on shoot growth or the concentration of P. With the brown earth soil, although the amount of infection was not increased, the introduced coarse endophytes had become established in the roots but no effect on shoot growth was observed in the year of sowing. However, in the first harvest year, in the presence of 4 kg P ha~', clover inoculated with Glomus etunieatus produced double the amount of dry shoot per hectare produced by uninoculated plants and 75 % greater than that produced with Glomus mosseae LI. These observations encourage the view that inoculation with mycorrhizal fungi of white clover for improved hill pastures might have a practical role if the responses could be made more predictable. It is concluded that further work is justified to understand the ecology of mycorrhizal fungi in hill pastures and how the symbiosis functions, to select suitable endophytes and to develop methods of inoculation appropriate for use in routine field practice. INTRODUCTION Since the early work of Baylis (1959), in which P was implicated in the enhanced growth of vesicular-arbuscular mycorrhizal (VAM) plants, studies have been directed towards the understanding and possible exploitation of this particuiar symbiosis. Daft and Nicolson (1966) suggested that the endophytic mycelium supplemented the absorbing system of the host. It is now generally accepted that plant growth is enhanced because fungal hyphae explore the soil outside the 5iutrient depletion zone and absorb ions, especially P, from the soil solution around Department of Biological Sciences, University of Dundee, Dundee DDl 4HN, UK.»28-646X/82/ S3./ 1982 The New Phytologist

2 go A. RANGELEY et al. roots and transfer them to the plant {Sanders and Tinker, 1971; Hayman and Mosse, 1972). Wastie (1965) commented on the ubiquitous nature of endomycor.- rhizas and pointed out that the potential benefits are already operating under field conditions. However, Daft and Nicolson (1966) suggested that the development of cultural techniques and manipulation of the fungal endophytes might prove advantageous, particularly when the host is both nodulated and mycorrhizal (Daft, 1978). White clover is an important component in improved hill pastures which must be established rapidly and without excessive cost, and managed to maximize both nitrogen fixation and impact on animal nutrition (Newbould and Haystead, 1978), For satisfactory establishment and growth of white clover in most hill soils, the soils must be limed and fertilized with phosphate. VA mycorrhizas, which are involved in phosphate nutrition, are common on the roots of grasses and clover, but in hill pastures the amount of infection naay be low (Sparling and Tinker, 1978a). Responses by white clover to inoculation with mycorrhizal fungi grown in hill soils have been recorded in glasshouse experiments (Powell, 1976; Sparling and Tinker, 1978b). Increased growth of white clover in the field after inoculation of seed and use of transplanted pre-infected seedlings has been reported by Powell (1977, 1979) in New Zealand. Similar responses occur in Welsh upland areas but only after transplanting pre-inoculated seedlings (Hayman and Mosse, 1979). Since the latter method is unlikely ever to be applicable on a farm scale, the use of inocula introduced at the time of sowing was investigated in the present study. The objective was to ascertain if there were potential benefits from the use of VAM with white clover in hill soils before establishing a more detailed study of possible endophytes and of techniques for field inoculation. MATERIALS AND METHODS Sites, soils, lime and fertilizers Two sites were selected for field experiments, one with a deep peat soil at the Hill Farming Research Organisation's Lephinmore Field Research Station, Strachur, Argyll, and the other with a brown earth soil at the Sourhope Field Research Station, Yetholm, Roxburghshire. Soils from these sites, for use in pot experiments in a growth room, were partially air dried before the peat was shredded and the brown earth sieved through a 2 nam mesh. The peat had a density of -1 g cm~', ph in water of 3-8 and 8 mg P per 1 g dry soil was extractable by acetic acid (Hende, Cottenie and de Johnghe, 1952); the brown earth had a density of -65 g cm~', ph of 4-4 with 6 mg P per 1 g dry soil. To create conditions suitable for the growth of white clover in these soils linae, phosphate (P) as superphosphate and potassium (K) as the chloride were added at the rates shown in Table 1. For the pot experiment, amounts equivalent to the field rates, calculated on the basis of surface area, were unifornaly mixed with the soil for each pot after sieving through a 25 /tm mesh sieve. Phosphorus (1 kg P ha~^, i.e. 44 mg per pot) was added to the soil in the pot experiment but not in the field to avoid a large gap in the range of values. The same volume of each soil was added to 75 cm diameter pots but, because of the different densities, the weights used were 31 g dry peat and 2 g dry brown earth. This meant that each pot of unfertilized deep peat soil had -25 mg and of brown earth 12 mg acetic acidextractable P. Thus, the lowest level of P fertilizer added was equal to the amount of extractable P in the brown earth soil and was 3 times more in the deep peat.

3 Mycorrhizal infection of clover g l Table 1. Quantities* of lime, phosphorus (P) and potassium (K) used in the pot and field experiments Pot (mg) Field (kg ha"') Lime Deep peat (DP) Brown earth (BE) 22 5 DP/BE DP BE 8' * Equivalent on a surface area basis, i.e. 33 mg linne per pot (45-6 cm^) = 7-5 t ha ^. A mixture of trace elements needed to ensure healthy growth and nitrogen fixation hy white clover was also applied, namely, in kg ha"-*, 1 CuSO,,, 2 CoSO^, 2 ZnSOj, -2 NaaMo4.2H2, 5 NajBp,, and on deep peat soils only, 1 MnSO^. Variations in these general conditions are noted under individual experiments. Hosts and endophytes Two clover cultivars were used. New Zealand Grasslands Huia and Aberystwyth S184. The rhizohial inoculum contained thtee strains, FA6, ldl and P3, from the East of Scotland College of Agriculture collection with 1 x 1'' bacterial cells per seed. Two species of mycorrhizal fungi from the Dundee collection were used: Glomus mosseae (Nicolson and Gerdemann, 1968) selection LI (El Giahmi, Nicolson and Daft, 1976) maintained on maize and Glomus etunicatus (Becker and Gerdemann, 1977) maintained on leek. The mycorrhizal inocula were used either as spores mixed into sterile sand or as finely chopped infected root segments and spores in sand. In the pot experiment, control plants received a filtrate containing other associated soil organisnns obtained from the mycorrhizal inoculum (Hayman and Mosse, 1971). Growth e-nvironments The controhed environment room had 15 C day/1 C night temperatures, 95 Wm^ photosynthetically active radiation (PAR) light for 12 h, with 9% humidity. The average clinaatic conditions at the field sites were: Mean annual Mean annual temperature rainfall CC) (range) (mm) Lephinmore 8-1 (Otol9) 216 Sourhope 73 (-1 to 17) 94 Assessme-nts Measurements were made of the production of dry matter by clover (shoots alone in the field and shoots and roots in the pot experiment). The roots were cleared and stained using the method of Phillips and Hayman (197) and the

4 92 A. RANGELEY et al. proportion of roots that were mycorrhizal was assessed using the Gridline intersect method (Giovannetti and Mosse, 198). In some instances, the proportion of infection caused by the fine indigenous endophyte, probably Glomus tenuis (Hall, 1977), and other coarser endophytes was also estimated. The phosphorus content of the shoots and roots was measured after digestion in sulphuric acid and hydrogen peroxide with addition of lithium sulphate and selenium (Parkinson and Allen, 1975) by colorimetric assay after production of a blue phosphomolybdate complex with SnCl^ (Allen, 1974). RESULTS Indigenous endophytes in the two soils To determine the number of viable propagules of indigenous endophytes, 1 g dry wt samples of each soil, collected from 5 to 1 cm depth on the same day in autumn, were mixed with previously autoclaved sand until the volume was sufiicient to fill four black plastic tubes (1 cm diameter and 7 cm long). The sand and soil were thoroughly mixed and three tubes were filled with the mixture. To the remainder, a further three volumes of sand were added and mixed. Thus, each dilution series contained tubes with 2-5 g, 625 mg, 156 mg, 39 mg and 1 mg of soil mixed evenly throughout sterile sand. There were three complete replicates of the dilution series for each soil and each series was placed together in a test-tube rack; the latter were completely randomized and placed in the growth chamber. Three seeds of white clover (New Zealand Grasslands Huia) were sown into the soil/sand mixture and were watered with half-strength nutrient solution minus phosphate ion (Dart and Pate, 1959). Drainage was through a small hole in the bottom of the tube. After germination, the seedlings were thinned to one per tube and grown for 4 weeks. The roots were then stained and examined for presence or absence of mycorrhizal infection. The densities of mycorrhizal propagules in the two soils were different (95 % confidence limits are shown in brackets). In the peat, there were '32 propagules g-i soil (-8 to 1-12) and, in the brown earth, 2-47 g^^ (1-28 to 4-75). When the different densities of the two soils are considered and the number of propagules per unit volume of soil calculated, the peat had -32 cm"^ (-8 to -112) and the brown earth had 3-8 cm"" (-83 to 3-9); the latter is more than 1 times greater than for the peat. The effect on growth and phosphorus content of white clover of inoculation with mycorrhizal fungi at five levels of added P in two hill soils in pots in the laboratory The effect of five concentrations of added P (, 2, 4, 1 and 2 kg P ha"^) with no inocula, or with either of the two VA endophytes was examined. Ten seeds of white clover (New Zealand Grasslands Huia) were sown on to the surface of the soil of each pot and 1-8 cm^ of sterile sand or sand containing only spores of G. mosseae LI or G. etunicatus were mixed with the seed into the top centimetre of the soil. Approximately 16 spores were added to each pot. After germination, the seedlings were thinned to three per pot (i.e. as near as possible to the number resulting in the field from 5 kg seed ha~^) and inoculated with a suspension of Rhizohium. The pots were placed in four replicated randomized blocks in the growth room and white clover was harvested after 8 weeks. Figure 1 shows the effect of the treatments on the dry wt and P concentration of the shoots and roots of white clover and upon the percentage of infection. Mean

5 Mycorrhizal infection of clover 93 Deep peat Brown eorth Z -Z - - I - ~ 7 I " Q- 5 = C! 4 ~ -3 S -2 -i 8 7 SO Gs r - I J ][ I In if if If if if if if ^ w r, f 4 Ii I / ZO Level of added P (kg P ha"') Fig. 1. The effect of inoculation with vesicular-arbuscular mycorrhizas on shoot weight and P content, root weight and P content, and level of root infection, of white clover grown in two hill soils at five rates of added superphosphate. Mean results for Glomus mosseae LI (LI) and Glomus etunicatus (Ge) are shown in the hatched columns. Least significant differences at P < -5 are shown. results for the two endophytes are given since in only one comparison (infection with no added P) which is indicated on the figure was there a significant difference between them although there was a trend at all concentrations of P in the brown earth soil for G. etunicatus to increase dry weight of shoots more than G. mosseae LI. Because of the wide range of shoot and root weights, the values (mg per three plants) were transfortned to natural logarithms for statistical analysis and the latter

6 94 A. RANGELEY et al (b) - -2 i TJ m gg r = -2 - l\ T 1 " 1 / / ^ O. 2 4 ioo 2 Level of added P (kg P ha"') IOO 2 Level of added P {kg P ha"') Fig. 2. The effect of added superphosphate on the total weight of root, the weight of mycorrhizal root and the P content of uninoculated roots of white clover grown in two hill soils; (a) deep peat, (b) brown earth., Percentage P;, total root;, mycorrhizal root. Least significant differences at P < '5 are shown. values are shown. Figure 2 records the effect of phosphate on the P content of uninoculated roots and on the weights of mycorrhizal and total root. Deep peat. In the peat, there was a large response in shoot dry weight to the addition of phosphorus. Inoculation with VA endophytes significantly increased production of dry matter with and 2 kg P ha-\ had no significant effect with 4 and 1 kg P ha"^ and tended to decrease growth with 2 kg P ha^^. In the uninoculated plants, the concentration of phosphorus in the shoot increased significantly with added phosphorus except between 2 and 4 kg P. Inoculation increased markedly the concentration of P in the shoot when and 2 kg P ha"^ were applied but had no effect at higher levels of added P. Root weight also increased with concentration of phosphorus and inoculation tended to increase it at all levels of added P although this was only statistically significant in the absence of added P. The concentration of P in the roots of uninoculated plants increased steadily from -12 to -25 % with increasing amounts of applied P but with inoculated plants remained about -22% up to lookgpha"' and then increased to -28 % at 2 kg P ha^^. Infection was 2 % or less in the uninoculated plants and inoculation significantly increased the percentage of infected root from 9 to 71 %. The percentage of infected root decreased as the level of phosphate increased, as did the weight of mycorrhizal root at levels greater than 4 kg P ha~^ [Fig. 2(a)]. Brown earth. In the brown earth, there was a small response in shoot dry wt to addition of P. Inoculation tended to decrease shoot weight at 2 kg P ha"'' (as in the deep peat soil) but, at all other levels of P, it tended to increase shoot weight and this effect was greatest with 4 kg P ha-^ Averaged over all phosphorus treatments, the shoots of plants inoculated with G. etunicatus were significantly heavier than those with G. mosseae LI (-58 compared to 44 mg dry wt per three plants) [L.S.D. (P < -5) = -12] although this was mainly due to the very low shoot weight found at 2 kg P ha^^ with the latter endophyte. In the uninoculated plants, the level of P in shoots was little affected by increased additions of P. Inoculation significantly increased P content at 4 kg P and tended to increase it in three of the four other comparisons.

7 Mycorrhizal infection of clover Q5 Weight of roots increased with added P although the response was not as great as in plants grown in deep peat soil [Fig. 2(b)], plants grown in the absence of P being larger in the brown earth soil. Inoculation had no clear effect on root weight although there was a trend for increase at the and 4 kg P ha"' levels. The concentration of P in the roots was little affected by added P but, at 4 kg P, inoculated roots had a significantly greater concentration of P than uninoculated roots. The overall mean concentration of P in the roots was -25 %. The percentage of roots infected with indigenous VA mycorrhizas ranged from 51 to 3% with the lowest infection occurring at 2 kg P ha~^. Inoculation had little effect on the amount of infection except at 4 kg P ha"'' where it caused a significant increase. However, a detailed examination of the type of infection (coarse or fine) showed a much higher proportion of the former type in all the inoculated plants (Table 2). Since the introduced endophytes are both of the coarse type, this indicates that inoculation had brought about colonization of root although the degree of infection was little altered. Table 2. The proportion of infection caused by endophytes with coarse hyphae after inoculation of white clover cv. Grasslands Huia with Glomus miosseae LI and Glomus etunicatus {both with coarse hyphae) in a brown -zarth soil in the pot experiment Amount of added phosphorus (kg P ha~*) Proportion of infection caused by coarse endophytes Uninoculated Inoculated* L.S.D. (P < -5) * Mean results for two endophytes. Influence of mycorrhiza, host and phosphate application on the growth of white clover in the field Deep peat soil. A comparison was made between the two cultivars of clover, given three levels of added phosphorus (, 2 and 2 kg P ha"^) and inoculated with G. mosseae LI. The peat was cleared of vegetation by cutting with an Allen scythe and the lime, fertilizers and trace elements listed in Table 1 were applied to the soil surface; no tillage was possible. White clover seeds (5 kg ha"^) coated with Rhizobium, together with the fungal inoculum, which consisted of chopped root segments mixed with sand (equivalent to 4 m^ ha"^), were broadcast in August 1977 on to the surface of the peat. There were three replicate plots for each treatment, each 2 x 2 m separated by a 1 m wide path. Samples of shoots and roots were collected 1 year later and Table 3 records the results discussed below. There was little growth of either cultivar in the absence of added P; at both levels of added P, SI 84 produced more dry wt of shoots than Huia. The lack of response to P in the latter cultivar probably reflects the high mortality of seedlings during the winter, particularly at 2 kg P ha"'. At all levels of P, the number of plants

8 96 A. RANGELEY et al. Table 3. Effect /Glomus mosseae LI and amount of phosphorus on shoot dry wt, the phospkom content of the shoot and on mycorrhizal infection in white clover cv. 's SI84 and Grasslands Huia, grown in deep peat in the field at Lephinmore for 12 months Cultivar Amount of added phosphorus (kg P ha">) Inocuium Shoot dry wt (kg ha"') Phosphorus in shoots (%) Mycorrhizai infection (%) S184 Huia L.S.D. (P < -OS) Glomus mosseae G. mosseae G. mosseae G. mosseae G. mosseae G. mosseae 3a* < la 373de 287cd 862e 877e < la la 129bc 116b 122bc 117b * The data for shoot dr>' wt were transfomned to log^ (x+i) for statistical analysis. Values are significantly different when the letter after the number is different. of Huia in June 1978 was at least one-third less than for SI 84; the numbers (S184, Huia) averaged for both mycorrhizal treatments at the three levels of added P were P (125, 25), 2 P (75, 2) and 2 P (76, 125). In neither cultivar nor at any level of P did inoculation have any significant effect on dry wt of shoots. The phosphorus content of clover shoots of both cultivars was increased by the higher level of added P (2) and, averaged over all treatments, inoculation with G. mosseae LI increased this content significantly (P < -5). With SI 84, the level of infection with indigenous fungi was about 15 % and this was unaffected by added P; inoculation with G. mosseae LI more than doubled infection. Mycorrhizal infection of Huia with indigenous fungi was similar to that in SI84 except that with 2 kg P it was 37 %. Inoculation with G. mosseae LI more than doubled the average infection level of the uninoculated plants. Brown earth soil. Huia and the endophytes, G. mosseae L! and G. etunicatus, were compared in this experiment at and 4 kg P ha~*. There were four replicates of each treatment. Lime and fertilizers were applied at the rates given in Table 1. The site was rotovated to destroy the native vegetation and cultivate the soil, and then rolled. White clover seeds at 5 kg ha"^ inoculated with Rhizobium, were sown in April 1978 immediately after broadcasting the fungal inocula in the same form and at the same rate used on the deep peat. To avoid desiccation, both seeds and inocula were raked into the surface layers of the soil. The shoots were sampled once in the year of sowing (October 1978) and twice in the subsequent year (July and October 1979). Roots were sampled in October 1978 and December Mycorrhizal infection in all root samples was recorded but it was only possible to discriminate between the type of endophyte in the uninoculated plants and those inoculated with G. mosseae LI. This was considered to be a reasonable conapromise since no significant difference between the behaviour of G. mosseae LI and G. etunicatus had been found in the pot experiment with this soil type (Fig. 1).

9 Mycorrhizal infection of clover 97 O I o o 6 o o "I «O O fn 11 "-a 5i g-.s s If o o ^ O B s 3 i.l 11 J 3 11 u I 4 C <!u a 6 V 5i a S K g > a

10 98 A. RANGELEY et al. Table 4 shows annual yields of dry matter and P content (%) of the shoots of white clover in 1978 and 1979, together with the amount of root infection. The number of plants at this site in June 1978 was greater than on the deep peat with no added P (8 plants m"**) for the three mycorrhizal treatments but less with added P (6 plants m"^^). In 1978 (the year of sowing), with both levels of added P, the yields of dry matter from the plants inoculated with G. mosseae LI were higher than the control whereas plants inoculated with G. etunicatus yielded less. However, the effects were not statistically significant because of the high variability. The concentration of P in the shoots averaged '34% and was unaffected by either level of superphosphate or inoculation. The high mycorrhizal infection in uninoculated plots (8% on average) was predominantly caused byfine eodophytes (69 % on average) with the remainder being coarse (12 %) (Table 5). Inoculation with G. mosseae LI (a coarse Table S. Effect of inoculation with Glomus mosseae LI and level of phosphorus on total mycorrhizal infection in white clover roots and that caused by fine and coarse endophytes : white clover cv. Grasslands Huia grown in brown earth soil at Sourhope, samples taken in year of sowing (1978) Level of infection (%) Inoculant Level of phosphorus (kg P ha"') Type of hyphae Fine Coarse Total* G. mosseae LI C mosseae LI L.S.D. {P<-5) The sum of the fine and coarse infection can be tnore than the total infection because both types of endophyte can occur in the same section of root. endophyte) gave almost the same amount of total infection (78 % on average) but a significantly greater proportion (36 %) of this was of the coarse type, suggesting the successful establishment of the introduced endophyte. The addition of 4 kg P significantly depressed the proportion of coarse endophyte in the inoculated roots (Table 5). Inoculation with G. etunicatus depressed infection although this was only statistically significant in the absence of added phosphorus (Table 4). In the first harvest year (1979), G. mosseae LI was without significant effect on yield although this tended to be greater than in the uninoculated plots. By contrast, G. etunicatus increased shoot weight significantly (P < -1) in the presence of added phosphorus and tended to decrease shoot weight where no P was added. In the latter case, the weight of shoot with G. etunicatus was significantly less than that with G. mosseae LI. The concentration of P in the shoots was unaffected by treatment and it averaged -27 %, being less than in the first year and more similar to the values found in the pot experiment (see p. 95). Mycorrhizai infection of roots was lower than in 1978, possibly owing to the later time of sampling and the loss of cortical tissue which contains most of the endophyte. However, infection

11 Mycorrhizal infection of clover 99 with G. etunicatus in the presence of added P was significantly greater than for the uninoculated plants and those inoculated with G. mosseae LI. The roots of plants inoculated with the latter endophyte grown in the absence of P were infected to the same level as with G. etunicatus and both were greater than for uninoculated plants. DISCUSSION The two soils represent the poorest (deep peat) and the best (brown earth) of the range of hill soil types assessed for potential productivity (Newbould, 1981) and they present very different problems for the establishment of introduced VAM endophytes on white clover in reseeded hill pastures. Mycorrhizal plants are rarely found in wet soils (Mejstrik, 1965) so that infection of uninoculated clover in the deep peat at Lephinmore was low (37 % or less). Despite the crude method of inoculation, the level of infection in clover was doubled but production of shoots was unaffected (Table 3). There was, however, an increase in. the phosphorus content of the shoots after inoculation; this common observation was discussed hy Stribley, Tinker and Rayner (198) who suggested that a greater concentration of phosphorus in mycorrhizal than non-mycorrhizal plants is caused by an increased demand for carbon from infected roots. Treatment of the peat and storage after collection may have killed propaguies of indigenous endophytes and would contribute to the very low level of infection in the uninoculated plants in the pot experiment (Fig. 1). These results agree with those of others who have measured the effect of mycorrhizal inoculation of white clover cv. Grasslands Huia in sterile soils. Hall, Scott and Johnstone (1977) and Crush and Caradus (198) found positive responses to mycorrhizal inoculation at the low end and negative responses at the high end of their treatments with phosphorus. However, with one endophyte {Glomus fasciculatus E3), Crush and Caradus (198) found that mycorrhizal plants produced greater dry wts over a wider range of applied P levels and Powell (198) also noted that two endophytes {Glomus tenuis and Gigaspora margarita) enhanced yield of white clover to near the maximum yield attained in uninoculated plants. Clearly, it is endophytes of the latter type which are required as inocula for agricultural crops. With the brown earth, in both pot and field, the introduced endophytes formed infections (Tables 2 and 5) hut total levels of infection with one exception (4 kg P ha"^ in the pot experiment) were not increased (Fig. 1). Moreover, clover plants responded only slightly to applied P (Fig. 1 and Table 5). Thus, the highly significant increase in shoot dry matter of clover inoculated with G. etunicatus when given 4 kg P ha"^ in the second year in the field experiment appears not to be mediated through enhancement of phosphorus nutrition. Chemical analysis of the shoots did not implicate a mycorrhizal effect on the N, K, Ca, Mg, Cu, Zn and S nutrition of the clover (unpublished observations) nor was there an effect of inoculation on the dry matter production from native grasses that were present as weeds in the plots. The development of a response in the second year of growth has been found hy Hayman and Mosse (1979) with pre-inoculated white clover seedlings transplanted on Welsh hills and also by Hall (pers. comm. 1981). It seems that introduced endophytes need time to establish before beneficial effects develop. In our field experiments, infection and colonization of introduced inoculum in roots of clover was successful and suggests little competition for infection sites between endo-

12 ioo A. RANGELEY et al. phytes. It seems that infection will readily take place from a propagule in the vicinity of the root. The information in Figure 2 suggests that specifically placed inoculum (at 1 cm depth) in soil may result in low levels of colonization by the introduced endophyte at moderate levels of soil P owing to the rapid growth of roots down through the soil and it may take several years for the inoculum to penetrate deeper into the soil and colonize a larger proportion of the root system^. The positive benefits of inoculation to the shoot growth of white clover in the deep peat soil occurred when a large part of the root system was mycorrhizal and the percentage P in the uninoculated non-mycorrhizal roots was -15 % or lower. In the hrown earth soil, inoculation had the largest effect on shoot weight (not significant) with 4 kg added P (Fig. 1) when the percentage P in the roots was -21 %, the lowest found in any treatment for this soil, and when the amount of mycorrhizal root formed 6 %, of the root weight. However, it was not possible to measure accurately the P concentration of non-mycorrhiza! roots grown in the brown earth because uninoculated roots were infected by indigenous endophytes. Thus, although the P content of plants varies with age and choice of tissue amongst other things (Bates, 1971), the present results fit with the view that responses to inoculation only occur when the P content of shoots is below a so-called ' critical' value; published values for this concentration in white clover range from -2 to -25 % (Andrew, 196; Jackman and Mouat, 1972; Rangeley, 198). Additionally, Mosse, Powell and Hayman (1976) with Trifolium pratense, Stylosanthes guyanensis and Centrosema pubescens, and Barea, Escudero and Azcon-G. de Aguilar (198) with Medicago sativa have shown that nodulation and nitrogenase activity were negligible when concentration of P in the plant was below -2%. Therefore, our data support the conclusions of Sanders and Tinker (1971) and Hayman and Mosse (1972) that mycorrhizas exert their eflect on growth by increasing the phosphate supply to shoots. Data derived from the pot experiment (Table 6) provide further evidence for this explanation since more phosphate was transported to shoots per grana of roots in the inoculated than the control treatments. Paradoxically, this effect was statistically significant {P < -5) overall for the brown earth but not the deep peat soil. Glomus etunicatus but not G. mosseae LI tended to benefit growth in the brown earth in the pot experiment and brought about a marked increase in growth in the field (Table 5). G. mosseae LI was isolated from a North African soil and G. etunicatus from a Scottish soil. The two endophytes affected growth equally in the pot experiment with the peat soil, so that, while adaptation to temperature may Table 6. The effect of mycorrhizas on the quantity of phosphorus {g) in shoots of white clover per unit weight {g) of total root in the pot experiment (mean results for all levels of added phosphate) Phosphorus in shoot (g g"' root) Deep peat Brown earth Uninocuiated Glomus mosseae LI 6'5 5"7 Glomus etunicatus L.S.D. (P < -5) -9-8

13 Mycorrhizal infection of clover have been a factor affecting performance in the field, there is some suggestion of an interaction with soil type. Mosse (1972, 1975) has stressed interactions between mycorrhizal endophytes and soil type and Lambert, Cole and Baker (198) found that indigenous endophytes do better than introduced endophytes in their own soil. However, since reseeded hill land requires large amounts of lime and fertilizers (Newbould, 1976), and som.e mycorrhizal populations change in effectiveness during pasture development (Crush, 1978), the introduction of an endophyte adapted to the changed conditions could obviate any adjustment time by indigenous endophytes. To be of practical assistance, the mycorrhizal treatment should either enhance establishment, early growth and nitrogen fixation by white clover in hill pastures or should lessen the requirement for added P. Our data suggest that, hy careful selection of endophytes with rapid colonization, the growth of white clover can be enhanced at moderate levels of P under field conditions. If achievable in a predictable manner, enhancementsof this type could become increasingly intportant as the cost of P fertilizer rises. Further work is needed on the ecology of indigenous endophytes, on the infection process, on the mechanisms by which the symbiosis is either beneficial or detrimental and on methods of field inoculation that can be used routinely in farm practice. loi ACKNOWLEDGEMENTS The authors are indebted to Mrs Julia Leask and Mrs Mary Thornton for technical assistance. RERERENCES ALLEN, S. E. (Ed.) (1974). Chemical Analysis of Ecological Materials, pp. 18^195. Blackweli, Oxford. ANDREW, C. S. (196). The effect of phosphorus, potassium and calcium on the growth chemical composition and symptoms of deficiency of white clover in a sub-tropicai environinent. Australian Journal of Agricultural Research, 2, BATES. T. E. (1971). Factors affecting critical nutrient concentrations in plants on their evaluation: a review. Soil Science, 112, BAREA, J. M., ESCUDERO, J. L. & AZCON-G. DE AOUILAB, C. (198). Effects of introduced and indigenous VA mycorrhizal fungi on nodulation, growth and nutrition of Medicago sativa in phosphate fixing soils as affected by P-fertilizers. Plant and Soit, BAYLIS, G. T. S. (1959). Effect of vesicular-arbuscular mycorrhizas on growth of Griselinia littoratis (Cornaceae). New Phytologist, 58, BECKER, W. N. & GERDEMANN, J. W. (1977). Glomus etunicatus sp.nov. Mycotaxon, 6, CRUSH, J. R. (1978). Changes in effectiveness of soil endomycorrhizal fungal populations during pasture development. New Zealand Journal of Agricultural Research, 21, CRUSH,]. R. & CAEADUS,,]. R. (198). EfTect of mycorrhizas on growth of some white clovers. New Zealand Journal of Agricultural Research, 23, DAFT, M. J. (1978). Nitrogen fixation in nodulated and myeorrhizai crop plants. Annals of Applied Biology, 88, DAFT, M. J. & NICOLSON, T. H. (1966). Effect of Endogone mycorrhiza on plant growth. New Phytologist, 65, DAHT, P. J. & PATE, J. S. (1959). Nodulation studies in legumes. III. The effects of delaying inoculation on the seedling symbiosis of barrelnriedic, Medicago trihuloides Desr. Australian Journal of Biological Science, 12, EL GIAHMI, A. A., NICOLSON, T. D. & DAET, M. J. (1976). Endomycorrhizal fungi from Libyan soils. Transactions of the British Mycological Society, 67, GIOVANNETTI, M. & MOSSE, B. (198). An evaluation of techniques for measuring vesicular-arbuscular mycorrhizal infection in roots. New Phytologist, 84, HALL, I. R. (1977). Species and mycorrhizal infections of New Zealand Endogonaceae. Transactions of the British Mycological Society, 68,

14 IO2 A. RANGELEY et al. HALL,, I, R., SCOTT, R. S. & JOHNSTONE, P. D- (1977). Effect of vesicular-arbuscular mycorrhizas on response of 'Grasslands Huia' and 'Tainar'' white clovers to phosphorus. New Zealand Journal of Agricultural Research, 2, HAYMAN, D. S.. & MOSSE, B. (1971). Plant growth responses to vesicular-arbuscular nnyoorrhiza. I. Growth of Endogone-moculatGd plants in phosphate-deficient soils. New Phytologist, 7, HAYMAN, D. S, & MOSSE, B. (1972). Plant growth response to vesicular-arbuscular mycorrhiza. HI. Increased uptake of labile P from soil. N'ew Phytologist, 71, HAYMAN, D. S. & MOSSE, B. (1979). Improved growth of white clover in hill grasslands by mycorrhizal inoculation, Annals of Applied Biology, 93, HENDE, A. VAN DEN, COTTENIE, A. & DE JOHNGHE, P. (1952). The value of some methods for chemical soil analysis applied to different Belgian soil types. Transactions of International Congress on Soil Science, n, IV, 2, JACKMAN, R. H..& MouAT, M. C. H. (1972). Competition hetween grass and clover for phosphate. I. Effect of Crowntop {Agrostis tennis Sibth.) on white clover (Trifolium repens) growth and nitrogen fixation. New Zealand Journal of Agricultural Research, 15, LAMBERT, D. H., COLE, H, JH & BAKEH, D. E. (198). Adaptation of vesicular-arbuscular mycorrhizae to edaphic factors. New Phytologist, 85, MEJSTRIK, V. K. (1965). Study on. the development of endotrophic mycorrhiza. In: Plant-Microbes Relationships. The Publishing House of C2echoslovakia Academy of Science, Prague. MossE^ B. (1972). The influence of soil type and Endogone strain on the growth of mycorrhizal plants in phosphate deficient soils. Revue D'Ecologie et de Biologie du Sol, 9, MOSSE, B. (1975). Specificity in VA mycorrhizas, In: Endomycorrhizas (Ed, by F. E. Sanders, B. Mosse & P. B. Tinker), pp Academic Press,, London, MOSSE, B., POWELL, C. L. SC HAVMAN, D. S, (1976), Plant growth responses to vesicular-arbuscular mycorrhiza, IX. Interactions between VA mycorrhizas, rock phosphate and symbiotic nitrogen fixation. New Phytologist, 76, NEWBOULD, P. (1976). Techniques for hiu land improvement used in the United Kingdom. In: Hill hands. Proceedings of Inter national Symposium 1976, West Virginia, pp NEWBOULD, P, (1981). The potential of indigenous plant resources. Occasional Symposium 12^ British Grassland Society pp NEWBOULD, P. & HAYSTEAD, A. (1978), Trifolium repens (white clover): its role, establishment and maintenance in hill pastures. Hill Farming Research Organisation^ 7th Report, pp, NrcoLSON, T. H. & GERDEMANN, J. W. (1968). Mycorrhizal Endogone species, Mycologia, 4, PARKINSON, J. A. & ALLEN, S. E. (1975). A wet oxidation procedure suitable for the determination of nitrogen and mineral nutrients in biological material. Communications in Soil Science and Plant Analysis, 6, 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 British Mycotogica! Society, 55, POWELL, C. LL. (1976). Mycorrhizal fungi stimulate clover growth in New Zealand hill country soils. Nature, 264, 436^38. POWELL, C. LL. (1977). Mycorrhizas in hill country soils,. III. Effect of inoculation on clover growth in unsterile soils. New Zealand Jfournal of Agricultural Research, 2, POWELL, C. LL. (1979). Inoculation of white clover and ryegrass seed with mycorrhizal fungi. Nezv Phytologist, 83, POWELL, C. LL. (198). Phosphate response curves of mycorrhizal and non-mycorrhizal plants. I. Response to superphosphate. New Zealand Journal of Agricultural Research, 23, RANGELEY, A. (198). The nutrient requirement of white clover on hill soils. Ph.D, Thesis, University of Edinburgh. SANDERS, F. E. & TINKER, P, B. (1971). Mechanism of absorption of phosphate from soil by Endogone mycorrhizas. Nature., 233, SPARLING, G. P. & TINKER, P. B. (1978a). Mycorrhizal infection in Pennine grassland. I. Levels of infection in thefield.journal of Applied Ecology, IS, SPARLING, G.F. & TINKER, P. B. (1978b). Mycorrhizal mfection in Pennine grassland. HI. Effects of mycorrhizai infection on the growth of white clover. Journal of Applied Ecology, 15, STRIBLEY, D. P., TINKER, P. B. & RATNER, J. W. (198). Relation of internal phosphorus concentration and plant weight in plants infected by vesicular-arbuscular mycorrhizas. New Phytologist, 86, WASTJE,, R. L. (1965). The occurrence of an Endogone type of endotrophic mycorrhiza in Heavea brasiliensis. Transactions of British Mycological Society, 48,

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