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 Sciences, University of Dundee {Received 18 April 1969) SUMMARY The effects of concentration of the inoculum on growth of the host and level of infection were investigated in mycorrhizal tomatoes. All concentrations of the endophyte, ranging between 225 and three chlamydospores per plant, led to significant stimulation of growth. Measurements of height of plants and length of leaves during growth indicated that inocula containing low numbers of spores stimulated the host less than more concentrated inocula. Plants inoculated with high numbers of spores produced more upper and retained more lower leaves whereas plants inoculated with low numbers retained more lower leaves than uninfected control plants. Dry weight determinations of the foliage made at the end of the experiment showed that all inoculated treatments were larger than the control treatments. The concentration of spores in the inoculum did not appear to influence the final level of infection. INTRODUCTION In previous experiments involving Endogone mycorrhiza (Daft and Nicolson, 1966, 1969) inoculation of plants was carried out at a standard level. The inoculum was usually adjusted so that each seedling received approximately fifty chlamydospores at transplanting and this always gave good infection. The inoculum consisted of fractions sieved from 'pot cultures' (Nicolson, 1967), which contained spores, mycelium and fragments of roots together with organisms which would colonize open pots in the glasshouse. In this laboratory over the past few years examination of roots at the initial stages of infection has shown that almost all the infections came from germinating spores and few from mycelium or infected root segments. The invasion and subsequent development of infection in roots by Endogone mycorrhizal fungi is likely to be affected by similar factors to those which influence root disease fungi, and these have been discussed by Garrett (1956). One of the factors would be the spore concentration in the inoculum or the 'inoculum potential' (Garrett, 1961). The experiment described here was conducted to examine the effects of concentration of the inoculum on the amount of mycorrhizal infection produced and the effects on host growth. Growth was measured throughout to determine the rate of host development in both mycorrhizal and control treatments and at the end dry weights were determined. 953

954 M. J. DAFT AND T. H. NICOLSON MATERIALS AND METHODS The endophyte used was Endogone macrocarpa var. geospora (Nicolson and Gerdemann, 1968). Six inocula were obtained by diluting a single stock culture containing a known concentration of chlamydospores. The number of spores in each concentration of inoculum was determined microscopically, using the method described previously (Daft and Nicolson, 1969). Tomato seed var. Eurocross, were germinated in sterile John Innes seed compost. Germination of seed and subsequent growth of the plants during the experiment was in a cellar converted as a growth room. Illumination was by means of fluourescent lamps (400 lumens/ft^ at plant height) controlled to give a daylength of 16 hours. The temperature was maintained at 20 C (±2 C). Selection of the seedlings and inoculation were as before (Daft and Nicolson, 1966). Plants were inoculated by pipetting 5 ml of the suspension, which had been examined microscopically to ensure that the requisite number of spores were present, into the hole made for the seedling plant. The plants were grown in sand culture and each received a twice-weekly application of Long Ashton nutrient solution at half the normal concentration. In this the potassium dihydrogen phosphate was omitted and phosphate was supplied in the form of bone meal (0.6 m-equiv./plant) which was mixed previously with the sand. The experiment consisted of two parts. In Part A, the growth of plants inoculated with 225, fifty-five and twenty-six spores per plant and a control treatment were compared. After 6 weeks growth, however, no conspicuous differences were evident amongst the three inoculated treatments. This was thought to be a result of the inocula being too concentrated. Hence, Part B was set up with the twenty-six-spore and control treatments repeated together with three other treatments of thirteen, seven and three spores per plant at inoculation. These lower spore concentrations were considered more likely to lead to differential effects amongst the treatments. Ten replicates were used for each treatment in Part A and Part B. Initially it was intended to measure growth of the tomato host throughout the experiment by continual assessment of leaf growth. This was used previously for mycorrhizal tobacco (Daft and Nicolson, 1966). While techniques for leaf measurement are available for tomato (Williams, 1954) these were found too time consuming and it was decided to use the more rapid measurements of plant height and leaf lengths recorded at weekly intervals. The sum of lengths of the internodes above the first foliage leaf gave the height for individual plants and from this the mean total height was obtained for each treatment. The length of leaves was obtained for individual plants by measuring each foliage leaf from the axil to the tip of the terminal leaflet. A mean total length of leaves was then calculated for each treatment. At the end of the experiment dry weights were obtained by drying to constant weight at 100 C. Only the foliage was used as roots were required for assessing the mycorrhiza. Phosphate contents were estimated as before (Daft and Nicolson, 1966). Assessment of the intensity of the mycorrhiza was made at the end of the experiment. In addition to percentage estimation, root infection was also assessed by grading the amount of yellow pigmentation present. This has been done for maize by Gerdemann (1964). Tomato is also a host which shows a distinct yellow colour in infected roots (Daft and Nicolson, 1966). In this experiment the roots were washed carefully at harvest and the amount of pigmentation estimated using an arbitrary scale of 1-9. This was done under a mercury vapour lamp which rendered the pigmented areas more easily visible.

Inoculum concentration of Endogone and growth 955 RESULTS Effect of infection on plant height and leaf length In Part A of the experiment growth was initially slow and it was not until some 20-30 days after inoculation that appreciable growth occurred (Fig. i). Changes in the rate of increase in plant height for all mycorrhizal treatments started 36 days after 30r 25 U r 20 1 10 5 (b) 0 90 80 70 I 60 > o 50 I 40 B 30 I 20 10 10 20 30 40 50 60 70 80 90 Days from inoculation Fig. I. Growth of mycorrhizal tomatoes inoculated with 225 (O), fifty-five ( ) and twenty-six (D) spores per plant; x, control (part A of the experiment), (a) Mean total height of mycorrhizal and control treatments; (b) mean total length of leaves of mycorrhizal and control treatments. inoculation. The first treatment to show a significant diflference (P <o.o5) from controls was the fifty-five-spore concentration at 44 days after inoculation (Fig. ia). Within the next 7 days all mycorrhizal treatments were significantly larger than the controls and remained so throughout the experiment. No statistically significant differences were found amongst the three inoculated treatments. The growth curves for leaf lengths showed a more rapid increase for inoculated plants than for controls (Fig. ib). This increase started 3 weeks from inoculation but statistically significant differences were

956 M. J. DAFT AND T. H. NICOLSON not evident until after 50 days. At that time the 225- and fifty-five-spore treatments were greater than controls and 7 days later the twenty-six-spore treatment also became significantly larger (P<o.o5). Again no difference occurred amongst the inoculated treatments. Part B of the experiment was set up later in the season, and growth during the initial 10 20 30 40 50 60 Days from inoculation 70 80 90 Fig. 2. Growth of mycorrhizal tomatoes inoculated with twenty-six (D), thirteen ( ), seven (A) and three (A) spores per plant; X, control (part B of the experiment), (a) Mean total height of mycorrhizal and control treatments; (b) mean total length of leaves of mycorrhizal and control treatments. Stages was more rapid than in Part A with increases evident some 25 days after inoculation (Fig. 2a). The height of plants with the highest spore concentration (twenty-six spores) became significantly larger than that of those with the lowest (three spores) and of the controls, after 62 days. A week later the seven-spore treatment was also significantly larger than the controls. Throughout the experimental period the two remaining treatments, the thirteen- and three-spore concentrations, did not become larger than the controls. The leaf length measurements showed that the twenty-six-spore treatment

Inoculum concentration of Endogone and growth 957 became significantly larger than the three-spore and control treatments at 40 days (Fig. 2b). The two treatments with thirteen and seven spores became significantly larger at 47 days and the lowest inoculum (three spores) significantly larger than controls at 58 days. These results show that the concentration of the inoculum did have an effect on the growth rate of the host. This is indicated by the times at which the various treatments became significantly different from the controls. In both parts of the experiment these times were progressively later with decreasing numbers of spores in the inoculum as far as leaf length measurements were concerned. Heights showed a similar trend though with some variability in that the fifty-five-spore treatment in Part A and the seven-spore treatment in Part B became statistically different earlier than the more concentrated 225- and thirteen-spore inocula respectively. Effect of infection on leaf development and retention The spatial distribution of leaves present on the inoculated treatments and their respective controls for both parts of the experiment are shown in Figs. 3 (a and b). In Fig. 3(a) it can be seen that the growth pattern of the infected and control treatments were similar until 28 days from inoculation. Seven days later the infected plants possessed on average one-and-a-half more upper leaves than the controls. This increase in the rate of leaf development continued and at the end of the experiment the 225-, fifty-five- and twenty-six-spore treatments possessed two-and-a-half, two-and-a-half and one-and-a-half more upper leaves than the controls respectively. Leaf fall in all four treatments started 34 days from inoculation. From this time an effect of infection was to reduce the rate of leaf fall. At harvest the 255-, fifty-five- and twenty-six-spore treatments retained on average two, two-and-a-half and one-and-a-half respectively more lower leaves than the controls. The results for part B (Fig. 3b) showed that the increase in the rate of leaf development started 27 days from inoculation for the twenty-six- and thirteen-spore treatments and 34 and 48 days respectively for the seven- and three-spore treatments. At the end of the growth period the twenty-six-, thirteen-, seven- and three-spore treatments possessed on average one-and-a-half, three-quarters, one and one more upper leaves than the controls. Leaf fall in all five treatments started approximately 30 days from inoculation. As found in Part A, the inoculated plants in Part B retained more lower leaves than the controls. When the experiment was terminated the plants in the twentysix-, thirteen-, seven- and three-spore treatments had retained two-and-a-half, three, two-and-a-half and one-and-a-half more lower leaves than the controls respectively. Although the two parts of the experiment were done at different times the three inoculated treatments in Part A and the twenty-six- and thirteen-spore treatments in Part B all show a marked similarity in the time when the increase in leaf number began (28 days for Part A and 27 days for Part B). With inocula below twenty-six spores per plant there is a progressive delay in the time of the increase in leaf numbers (Fig. 3b). Effect of inoculum concentration on dry weight, phosphate content, root infection and pigmentation The plants in both parts of the experiment were harvested after 12 weeks growth and the relevant data are given in Table i. The dry weights of foliage for all the mycorrhizal treatments were significantly larger (F<o.o5) than their respective controls. However, there were no statistical differences amongst the mycorrhizal treatments in

0 10 ZO 30 40 50 60 70 Days from inoculation 10 20 30 40 50 Days from inoculotion Days from inoculation Fig. 3. Effects of inoculum concentration on apical leaf development and basal leaf retention in mycorrhizal and control tomatoes, (a) Plants inoculated with 225 (o), fifty-five ( ) and twenty-six (D) spores per replicate (part A of the experiment), (b) Plants inoculated with twenty-six (D), thirteen ( ), seven (A) and three (A) spores per replicate (part B of the experiment). The upper curves on each graph represent the limits of apical leaf development and the difference between mycorrhizal (symbols) and control (x) plants represent the extra leaves developed by the former. The lower curves on each graph represent the differences in leaf retention at the basal internodes of mycorrhizal (symbols) and control (x) plants. Thus, the hatched areas between the graphs represent the extra leaf development and the stippled areas the extra leaf retention of the mycorrhizal plants compared to the respective nonmycorrhizal control plants.

Inoculum concentration o/endogone and growth 959 either Part A or Part B of the experiment. Hence, the differences amongst the treatments for height and leaf measurements found during the experiment were not seen in the dry weights at the end. As had been found previously (Daft and Nicolson, 1966) phosphate contents of the mycorrhizal plants were higher than in the control treatments. The figures for percentage infection were not related to either the numbers of spores in the inoculum or the dry weights, but the pigmentation figures showed some relationship to dry weight. In Part A of the experiment the fifty-five-spore treatment gave the highest mean rating and produced the largest plants. In Part B the three-spore treatment produced the smallest plants and was assigned the lowest pigmentation rating. Table i. Dry weights, phosphate contents, percentage infection and pigmentation ratings for tomatoes inoculated with six different spore concentrations (plants grown for 12 weeks) Experiment Part A Part B Treatment (spore no./ plant in inoculum) Control 225 55 26 Control 13 Mean dry weight of foliage (g) 0.182 (±0.07) 0.627* (±0.23) 1.05* (±0.38) 0.63* (±0.24) 0.308 (±0.11) 0.868* (±0.38) 0.908* (±0.25) 0.938* (±0.34) 0.688* (±0.24) Total phosphate Mean content of infection foliage (%) (// M/ioo mg dried material) 3-6 42-3 3-9 33-6 3-2 36.2 2.4 3-2 43-6 3-0 45.4 4-2 49.6 3.6 44.7 * Significantly different from control treatments at 5% level of prohability. DISCUSSION Mean pigmentation rating (arbitary scale 1-9) Continuous infection throughout the root system rarely occurs with phycomycetous mycorrhizas. They are usually sporadic and localized and furthermore, individual infection areas may not encircle the cortex. Typical distributions of infection both within a root system and in a root were clearly illustrated in the early work of Janse (1896). Amounts of infection vary greatly in the field and under experimental conditions we have found that it can be as high as 90% or as low as 25% with different phosphate sources and concentrations. Also infection may vary in different experiments using the same level of phosphate nutrition. Varying degrees of host stimulation can also be found (Daft and Nicolson, 1966). These factors suggested that the initial concentration of the infective propagules, in this case spores, might affect the growth of the host differently by inducing different intensities of infection. Measurements of heights of plants and lengths of leaves gave a good assessment of growth showing when differences between mycorrhizal and control plants occurred (Figs. I and 2). It was not possible to determine the stage and extent of infection when growth was first stimulated as this would have necessitated destructive harvests. The 4.2 5-7 S-o 4-3 4.2 4.6 3.8

960 M. J. DAFT AND T. H. NICOLSON most important events for stimulation would seem to be how soon infection takes place and how quickly it spreads within the root system. Examination at the end of the experiment (Table i) only shows how far infection has developed and may not reflect what has happened previously. Treatments containing high numbers of spores in the inoculum showed differential growth rates but when the inoculum was reduced to twenty-six spores or less the differences were more marked. These differences showed themselves by the times at which the treatments became significantly larger than controls. In general, these times were delayed with decreasing numbers of spores in the inoculum. Estimates for percentage root infection were not closely related to the dry final weight figures. Those for pigmentation bore a closer relationship and it may he that this is a more reliable indication of infection levels. Expression by percentage infection has been criticized (Gerdemann, 1968) and a later paper (Daft and Nicolson, in preparation) will examine more closely methods for quantitative assessment of mycorrhiza. The most striking influence of mycorrhiza shown here is on the pattern of plant growth. When the host is inoculated with a high concentration of spores more apical leaves are developed and more lower leaves are retained (Fig. 3a). With very low numbers of spores the development of leaves at the apex is similar to that of the urdnoculated control plants, but the number of basal leaves retained is greater (Fig. 3b). Hence, the results show that mycorrhiza stimulates the ontogeny and delays the senescence of leaves. These effects may be due to the production and regulation of hormones which are important in both development (Humphries and Wheeler, 1963) and senescence (Osborne, 1967) of leaves. Alternatively, the effects may be nutritional as in a mycorrhizal plant there is enhanced uptake of ions with more nutrients for internal redistribution. In the experiment reported here, phosphate analysis of all treatments in both Part A and Part B showed higher contents in the mycorrhizal plants (Table i). Hopkinson (1966) considers that leaf senescence to some degree is controlled by the availability of nutrients, such as phosphorus, from the soil. He recognizes four stages in the ontogeny of a leaf. The second of these is a period of rapid expansion which is associated with a high rate of mineral nutrient uptake into leaf tissues. In the third stage leaf growth declines with the concomitant export of phosphorus (Hopkinson, 1964). The chlamydospores of the Endogone species used in this investigation are large and have considerable food reserves for germination. This capacity for development of high infections from low concentration of spores may be very important in the field where populations are low (Nicolson, 1967; Mosse and Bowen, 1968). However, experimental conditions and those found in natural soil are considerably different. In the latter, the inoculum would be scattered throughout the soil giving many infection foci. In our experiments, the inoculum was not distributed throughout the sand but was placed in the hole made for the seedling plant. In this way the roots grew through the inoculum and the subsequent spread of the infection must have been by the endophyte growing along the roots and through the sand. A high level of infection from an inoculum of only three spores arose in an annual host within 12 weeks with considerable enhancement of growth (Table i). In mycorrhizal perennial hosts with yearly growth increments, such as those examined by Baylis (1967), the benefit accrued would be proportionately greater. It would appear then that high levels of infection may arise from a low inoculum. Applying terms used for plant pathogenic fungi (Garrett, 1961), very low numbers of Endogone spores, indeed even one spore, may constitute 'effective inoculum' with

Inoculum concentration 0/Endogone and growth 961 'independent' rather than 'synergistic' action. Garrett also suggests that the 'invasive force which the inoculum must supply is likely to reach its minimum values in symbiotic host-parasite relationships'. This would be particularly true for vesieular-arbuscular endophytes which, though highly adapted obligate parasites (Mosse, 1963), display a surprising lack of host specificity. As has been suggested previously (Nieolson, 1967), the answer to many of the unusual features regarding these mycorrhizas may be that the endophytes have evolved collaterally with the evolution of the land flora. There is convincing evidence (Butler, 1939) that similar infections were present in the earliest of vascular plants. ACKNOWLEDGMENTS We are grateful to Professor J. L. Harley, F.R.S. for reading the manuscript. We are also indebted to Miss E. Davidson for assistance with the figures and to Miss R. Kett and Mr S. Petrie for technical help. REFERENCES BAYLIS, G. T. S. (1967). Experiments on the ecological significance of phycomycetous mycorrhizas. New Phytol, 66, 231. BUTLER, E. J. (1939). The occurrences and systematic position ofthe vesieular-arbuscular type of mycorrhizal fungi. Trans. Br. mycol. Soc, 22, 274. DAFT, M. J. & NICOLSON, T. H. (1966). Effect of Endogone mycorrhiza on plant growth. I. New Phytol 65, 343- DAFT, M. J. & NICOLSON, T. H. (1969). Effect oi Endogone mycorrhiza on plant growth. II. Influence of soluble phosphate on endophyte and host in mycorrhizal maize. New Phytol., 68, 945. GAHSETT, S. D. (1956). Biology of Root-infecting Fungi. London and New York. GARRETT, S. D. (1961). Inoculum potential. In: Plant Pathology: An Advanced Treatise. Vol. III. The Diseased Population Epidemics and Control (Ed. by J. G. Horsfall & A. E. Dimond), Chapter 2. New York and London. GERDEMANN, J. W. (1964). The effect of mycorrhiza on the growth of maize. Mycologia, 53, 254. GERDEMANN, J. W. (1968). Vesieular-arbuscular mycorrhiza and plant growth. A. Rev. PL Path., 6, 397. HoPKiNSON, J. (1964). Studies on the expansion of the leaf surface. IV. The carbon and phosphorus economy of a leaf. jf. exp. Bot., 15, 125. HOPKINSON, J. (1966). Studies on the expansion of the leaf surface. VI. Senescence and the usefulness of old leaves, y. exp. Bot., 17, 762. HUMPHRIES, E. C. & WHEELER, A. W. (1963). The physiology of leaf growth. A. Rev. PL Physiol., 14, 385. JANSE, J. M. (1896). Les endophytes radicaux de quelques plantes Javanaise. Annls jfard. bot. Buitenz., 14. 53- MOSSE, B. (1963). Vesieular-arbuscular mycorrhiza: an extreme form of fungal adaption. Symp. Soc. gen. MicrobioL, 13, 146. MOSSE, B. & BOWEN, G. D. (1968). The distribution of Endogone spores in some Australian and New Zealand soils, and in an experimental field soil at Rothamsted. Trans. Br. mycol. Soc, 51, 485. NICOLSON, T. H. (1967). Vesicular arbuscular mycorrhiza a universal plant symbiosis. Sci. Prog., Oxf., 55. 561. NICOLSON, T. H. & GERDEMANN, J. W. (1968). Mycorrhizal Endogone species. Mycologia, 60, 313. OsBOKNE, D. J. (1967). Hormonal regulation of leaf senescence. Symp. Soc. exp. Biol., 21, 305. WILLIAMS, R. F. (1954). Estimation of leaf area for agronomic and plant physiological studies. Aust. J. agric Res., 5, 235.