EFFECT OF ENDOGONE MYCORRHIZA ON PLANT GROWTH

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1 EFFECT OF ENDOGONE MYCORRHIZA ON PLANT GROWTH BY M. J. DAFT AND T. H. NICOLSON Department of Botany, Queen's College, Dundee {Received 3 January 1966) SUMMARY The growth effects of three mycorrhizal Endogone endophytes on tobacco, tomato and maize were examined. The extent of the stimulus obtained depended on nutrient conditions and the subsequent level of infection developed in the root system. Marked stimulation occurred with low phosphate availability and high root infection. INTRODUCTION Evidence increasingly shows that Phycomycete or vesicular-arbuscular mycorrhiza may, under certain conditions, considerably affect the growth of the higher plant. This has recently been discussed by Gerdemann (1964). As for ectotrophic mycorrhiza (Harley, 1959, 1965), increased growth with Phycomycete mycorrhizal infection has been associated with enhanced nutrient uptake, particularly of phosphate (Baylis, 1959; Holevas, 1964; Gerdemann, 1965). Also it is now evident that a number oiendogone species may induce this type of mycorrhiza. These can be identified on the basis of sporocarps (Mosse, 1956; Gerdemann, 1961; Koch, 1961; Stevenson, 1964), large soil-borne spores (Gerdemann, 1955, 1961; Gerdemann and Nicolson, 1963) and on other features such as the type of root infection (Koch, 1961; Meloh, 1963). The aims of the work reported here were firstly, to assess the effects of three Endogone species on different hosts, and secondly, to investigate the influence of varying nutrient conditions, especially phosphate, on the mycorrhizal response. METHODS The endophytes used were three Endogone species originally obtained from soil in the Dundee area and designated spore types i, 2 and 3 (Gerdemann and Nicolson, 1963). Further studies of these spore types showed that they were distinct, the No. i spore type being sporocarpic and identical with one reported from Illinois (Gerdemann, 1961). The Nos. 2 and 3 spore types were non-sporocarpic species, differing in size and colour of spores and in some details of their life cycles (Nicolson and Gerdemann, 1962). Because Endogone species cannot be grown satisfactorily in artificial culture the endophytes have been maintained on host plants grown in sterile soil or sand. Inocula for experiments were obtained by wet-sieving such 'pot cultures' and storing the sieved fractions in water at 4 C. This consisted of material passing through a 420 pi mesh sieve hut retained on one of a 150 /i mesh size and contained root fragments, spores and hyphae of the endophytes. In the case of the No. i species sporocarps were also present. While infection of roots could come from any of these sources it was found that most were caused by germinating spores. The three principal hosts used were the F^ hydrid tomato var. Eurocross 'A', tobacco 343

2 344 M. J. DAFT AND T. H. NICOLSON var. White Burley and a single cross maize hydrid (kindly supplied by Professor J. W. Gerdemann, University of Illinois). Some preliminary experiments were made using the tomato var. Ailsa Craig. The plants were grown in a sand mixture which consisted of 2 parts washed sand collected from an embryo dune zone and i part of a coarse horti.. cultural sand. The dune sand was used as this had been noted previously to be suitahle for establishing infection (Nicolson, i960). The sand mixture was sterilized by autoclaving (15 lb pressure for 40 minutes). A weighed amount of sand was placed in each pot (4 or 5 in. size). The tomato and tobacco seed were germinated in sterile John Innes seed compost and the maize in washed sterile sand. When the seedlings were large enough to handle they were selected for uniformity and the roots washed free of the medium. The plants were inoculated by pipetting 4 ml of a suspension containing approximately fifty spores into the hole in the sand made for the seedling plant. The stock inoculum was kept stirred to keep the spores suspended. This method of inoculation ensured that the roots grew through the inoculum. plants received an equivalent fraction taken from nonmycorrhizal plants. This ensured that the microbiological components were similar, except for the presence of the endophyte, in both control and mycorrhizal treatments. In most experiments at least ten replicates were used for each treatment. During the growing season plants were grown in a glasshouse and in winter months in glass incuhators (Daft, 1965) with 13 hours daylight and at a temperature of 18" C. Each pot received a nutrient solution (50 ml/week) made up from the standard stock solutions descrihed hy Hewitt (1952, p. 189) but at half the normal concentration. The standard phosphate solution was omitted from the experiments in which phosphate was supplied to the plants in a solid form. The comparative effects of the endophytes on plant growth were measured hy the following: fresh weight, dry weight (oven dried to constant weight at 100 C), leaf area and total phosphate content of roots and leaves. The total phosphate content was estimated by the isobutanol-benzene method. The infection level developed in inoculated roots was estimated by the 'root slide' method (Nicolson, i960). Five slides each with 100 root segments from a number of plants were usually recorded to obtain a mean percentage infection for one treatment. Of the data collected only the growth measurements were analysed statistically. Inoculated plants were always checked to see that the treatment had been effective and the control plants examined to ensure that no accidental infection had taken place. This was easier with maize and tomato where infected roots produce a distinct yellow colour. RESULTS Effect of infection in the presence of a completely available inorganic nutrient supply Tobacco plants were inoculated with the three endophytes and the area of each growing leaf was measured at intervals. Table i shows that infection with the three endophytes increased the leaf areas. This increase was more apparent up to 60 days from inoculation than at the end of the experiment. Analysis did not show any interaction amongst the leaves of different ages and infection; this may indicate that where the leaf areas were greater the whole plant was larger than the controls. In another experiment, again using tobacco, the influence of a non-sporocarpic endophyte (No. 2) was determined by harvesting plants every 2 weeks from inoculation and then weighing the whole dried plant (Fig. i). When analysed together the second, third, fourth and fifth harvests showed a highly significant difference between the treatments

3 Mycorrhiza and plant growth 345 and that there was no interaction of time against treatment. This lack of interaction and the shape of the curves suggests that the effect of infection is accumulative. The final harvest was taken 70 days after inoculation and this corresponds approximately to the period of greatest increase found in the leaf area measurements (Table i). Tomato Table i. Leaf areas and infection levels of tobacco infected with three Endogone endophytes {Plants supplied vxith complete nutrient solution) Days after inoculation Mean Endophyte Mean leaf area (cm^) infection No. I (sporocarpic) No. 2 (non-sporocarpic) No. 3 (non-sporocarpic) * 11.7* * 42.4* 33-O 79.1* 83.1* * Significantly different from controls at 5 o level IO plants (var. Ailsa Craig) infected with the three endophytes and dried 66 days after inoculation showed that only endophyte No. i had significantly increased the final dry weights compared to the controls (Table 2). Time (weeks) Fig. I. Growth of tobacco plants infected with endophyte No. 2. ( ); endophyte No. 2 ( ). The infection in the tobacco roots varied somewhat with the three endophytes (Table i), and all showed extensive secondary mycelium and chlamydospores had formed. The tomato plants (Table 2) were harvested earlier than the tobacco and the endophytes had not produced the chlamydospore stage. The data presented in this section shows that when all nutrients were supplied in an available form some growth stimulus could be found with each endophyte but the effects were variable and were not as marked as those reported by other workers (Gerdemann, 1964; Baylis, 1959). N.P. F

4 346 M. J. DAFT AND T. H. NICOLSON Effect of infection as influenced by bone-meal as a phosphate source Sodium dihydrogen phosphate, which is the phosphate salt in the standard nutrient solution was omitted and various quantities of bone-meal were substituted. Two experiments were carried out using tomato as the host a preliminary one with three bone-meal levels and a second involving six levels. Results for the first experiment are given in Table 3. Pots at level i contained bone-meal equivalent in phosphate content to the total inorganic phosphate as supplied in the complete nutrient solution. This gave an initial phosphate content of 1.2 milligram-equivalents per pot. Differences between control and mycorrhizal treatments in all the three levels of honemeal were marked, being greatest at the lowest phosphate level and least at the highest. The degree of mycorrhizal infection varied, the highest infection occurring at the lowest phosphate concentration (Table 3). Table 2. Dry zveights and infection levels of tomato {var. Ailsa Craig) infected with' three Endogone endophytes {Plants grown for 66 days and supplied with complete nutrient solution) Mean dry Mean Endophyte weight (g) infection (tops) (%) No. I 1.789* 54.4 No No I-52 - * Significantly different from controls at 5 o level. Table 3. Drv zveights and infection levels of tomato {var. Ailsa Craig) grown in three bone-meal concentrations and infected with endophyte No. 1 {Plants grozvn for 76 days and supplied zvith nutrient solution minus the phosphate salt) Relative _,, -, ^ \ Mean phosphate Mean dry weight (g) infection level mycorrhizal ( o) ** ** Significantly different from controls at i o level. In the second experiment the Fj hybrid Eurocross was used to reduce the experimental variation found previously with the Ailsa Craig variety. The plants were grown in the glass incubators during the winter months and for practical reasons it was not possible to set up all the treatments at one time. Hence the experiment was divided into two sections A and B. Level 2 was replicated in both series and there was a difference in planting time of I week. Again there was a marked difference between the mycorrhizal and control treatments over most of the phosphate levels particularly the lower ones (Table 4). The infected plants developed larger and more leaves and the numbers of internodes were greater than those found on the control plants (Plate i8a). The control plants at the lower levels of bone-meal showed marked symptoms of phosphate deficiency as seen in the leaf colour and premature drop of cotyledons. At all levels the leaves and roots of mycorrhizal plants contained higher total phosphate than control plants (Table 4). At level 2 in the first series (A) the treatments were not significantly different but in the

5 THE NEW PHYTOLOGIST, 65, 3 PLATE 18 (a) Tomato plants fvar. Eurocross) grown in three levels of bone-meal. M. mycorrhizal; C, control plants, (b) Maize plants grown with bone-meal as phosphate source (level i). C, control; i, 2 and 3, mycorrhizal plants infected with the three separate endoph\tes. M. J. D.\FT AND T. H. ys^mycorrhlza AND PLANT GROWTLI {facing page 346)

6 THE NEW PHYTOLOGIST, PLATE 19 TPI CALClUVt PHOSPHATE Tomato plants (var. Eurocross) Krnwn: (a) with bonu-nieal as phosphate source (level i); (b) in various phosphate sources (le\el i ). C, control; M, niycnrrhizal; i. 2 and 3, mycorrhizal plants infected witll the three separate endophytes. M. J. DAFT AM, T. H. mco\.so\ MYCORRHIZA AND PLANT GROWTH

7 Mycorrhiza and plant groivth 347 same level in the second series (B) mycorrhizal plants had significantly higher dry weights than controls but only at 5% probability level. In the phosphate levels 4 and 8 there was no significant difference between mycorrhizal plants and controls. Hence from these two experiments it is evident that the mycorrhizal condition was more effective in increasing plant growth at low levels of phosphate nutrition. With higher levels the differences were less marked and at the highest level only slight stimulation was found. Similar results have been noted for strawberry (Holevas, 1964). Table 4. Dry weights, total phosphate contents and infection levels of tomato {var. Eurocross) grown in six bone-meal concentrations and infected with endophyte No. i {Plants grown for 65 days and supplied with nutrient solution minus the phosphate salt) Relative phosphate level I Mean dry weight (g) 0.393** ** ** s 0.516* Total phosphate content ifimlioo mg dried plant material) Roots Leaves **, * Significantly different from controls at i",j and 5",j levels respectively. Mean infection The greater total phosphate content found in infected plants both in roots and leaves shows that the association of the fungus and the plant root system induces more phosphate to be taken up. Even where the dry weight difference was negligible the phosphate content was higher. Root infection of plants grown at phosphate level 0.25 was 90.5% and at level 8 was 61.4%. The intermediate levels of phosphate gave progressively lower infection ratings with increasing phosphate content (Table 4). The degree of infection from level 2 in Section A was 63.9% and from level 2 in Section B, 73.4%. This higher rating may explain the statistical results found at level 2. Having established the general level of phosphate at which conspicuous differences between mycorrhizal and control plants could be obtained further experiments were carried out with the three endophytes on different hosts. The results for maize (Plate 18b) and tomato (Plate 19a), grown in bone-meal at level i, are shown in Table 5 and it can be seen that infection with each endophyte significantly increased the final dry weight. There did not appear to be any consistently different effects amongst the three endophytes and it is probable that they act similarly with perhaps only minor differences with different hosts. With maize the fresh and dry weights were significantly different. Both shoot and root contributed more or less equally to the final dry weight. ("0)

8 348 M. J. DAFT AND T. H. NICOLSON Effect of infection as influenced by three calcium phosphate salts Further investigations tested the influence of three phosphate sources on the mycorrhizal response (Plate 19b). Finely-ground apatite, tricalcium and dicalcium phosphate were used at concentration i. These compounds gave a range of phosphate availabilities, the availability of the tricalcium phosphate being intermediate between apatite and the dicalcium salt. The greatest difference between infected and control plants Table s. Dry zveights and infection levels of tomato, and fresh weights, dry zveights and infection levels of maize grozvn in bone-meal {level i) and infected zvith three Endogone endophytes {Plants supplied with nutrient solution minus the phosphate salt) Endophyte No. I No. 2 No. 3 Tomato (var. Eurocross) (harvested after 69 days) Mean total dry weight (g) 1.^7** i.:;i** O.42 Mean infection Co) _ Maize (hydrid) (harvested after 56 days) Mean total Mean dry Mean fresh weight weight (g) infection (g) Shoot Root (%) 38.7** 2.148** 1.998* ** 2.224** 1.763* ,8** 2.238** 1.968* O-975 **, * Significantly different from controls at i o and 5% levels respectively. Table 6. Dry zveights and infection levels of tomato {var. Eurocross) grown in three different phosphate sources {level i) and infected with endophyte No. i {Plants grozvn for 66 days and supplied with nutrient solution minus the phosphate salt) Mean Mean Mean Phosphate source dry weight dry weight infection (g) (g) (%) Apatite ** 76.6 Tricalcium phosphate ,67** 51.4 Dicalcium phosphate ** 24.6 ** Significantly different from controls at 1% level. occurred with the tricalcium salt (Table 6). This may indicate that the availability of phosphate is of importance, as the highest degree of infection was found in the apatite treatment. Of the plants kept for 5 weeks after the main harvest, only infected plants grown in the dicalcium phosphate had flowered and set fruit. DISCUSSION While a majority of workers have found increased growth of the host with Phycomycete mycorrhizal infection (Baylis, 1959; Winter & Peuss-Schonbeck, 1963; Clark, 1963; Stevenson, 1964; Gerdemann, 1964, 1965), others have not been able to show any such growth effect either experimentally (Thornton, ; Wastie, 1965) or under field conditions (Otto, 1962). Indeed, others have considered that the condition may he pathogenic (Jones, 1924; Wilhelm, 1959). The present observations stress the importance of the two related factors of nutrient availability and level of root infection in obtaining consistently high and significant differences between mycorrhizal and non-mycorrhizal plants. This supports the general previously held view that maximum host benefit and

9 Mycorrhiza and plant growth 349 infection occur only when conditions are least favourable for plant growth (Mosse, 1963)- In general, where the nutrient regime was high and all nutrients were supplied in a readily available form it was difficult to obtain growth increases with mycorrhizal infection. It was only with low conditions of phosphate availability that conspicuous differences could be shown. Here, root growth was reduced and this might be of importance as the establishment of infection may take up to 3 weeks. This slower root growth may also allow greater development of infection in the root system. Low phosphate regimes induced more marked deficiency symptoms in control than mycorrhizal plants (Plate 18a), particularly in tomatoes where premature cotyledon and leaf fall occurred only in control plants. Using soils with low phosphate content Gerdemann (1964) found that non-mycorrhizal maize plants developed symptoms of phosphorus deficiency. The total phosphate content of tomato roots and leaves (Table 4) indicates that the mycorrhizal root took up more phosphate from a bound source. From his experiments with Griselinia Baylis (1959) suggests that enhanced phosphate uptake is a favourable effect of mycorrhizal infection. The accumulative effect of infection found in tobacco (Fig. i), is in agreement with the results of Clark (1963) who found that with Liriodendron tulipifera L. the stimulatory effect of an endophytic infection was not immediate. In most cases when highly significant growth differences between mycorrhizal and control plants were evident, the root systems were infected in excess of 50/0. With conspicuous differences (Table 4) the infection level was up to go% and when smaller differences occured the infection levels were lower. However, high infection is not the only factor involved when considering the mycorrhizal response. This can be present with no detectable growth difference (Table 4) and conversely a low infection level can sometimes induce marked growth stimulation (Table 6). The results indicate that the effects of mycorrhiza on plant growth depend to some degree on the balance between the development of root infection and the concentration and availability of phosphate and perhaps other nutrients. Few quantitative data are available on amounts of root infection in nature. In apple, Mosse (1959) recorded almost complete infection of certain categories of roots and Otto (1962) found almost 100% infection after 3 years growth but the degree of infection was inffuenced by such factors as season and manurial treatments. With grass species from non-agricultural communities, levels of infection greater than 45% were rarely noted (Nicolson, i960), but in pasture grasses over 80% infection has been recorded after 30 weeks growth (Gadgil, 1965). These and other reports indicate that in nature infection frequently reaches a level potential for host stimulation. As has been pointed out (Wastie, 1965), the ubiquity of mycorrhiza under field conditions suggests that any possible advantage of its presence is already operating. However, by manipulation of the nutrient conditions or the prevalent endophyte species it may be possible to increase any stimulus already present. It has been suggested that the mechanism for any host stimulation with mycorrhiza may be highly specific such as the secretion of a growth-promoting substance (Butler, 1939; Otto, 1963). However, it may be that the endophytic mycelium on and surrounding the roots acts as a general absorbing system supplementing that of the hosts' roots. In all the experimental material we examined there was profuse mycelium in the rhizosphere and on root surfaces, with numerous connections to mycelium in root tissues and potentially the endophytes could act in this way. Such a system would confer greater relative benefit to the host under poor nutrient conditions. The universal occurrence of

10 35 M. J. DAFT AND T. H. NICOLSON these types of endophytes suggests that the mycorrhizal root may be the association concerned in the uptake of nutrients from soils of low fertility. ACKNOWLEDGMENTS We wish to thank Professor J. L. Harley, F.R.S., for reading the manuscript; Miss D, E. A. Strevens for statistical advice and Mr. A. Machar for technical assistance. REFERENCES B.^YLis, G. T. S. (1959). Effect of vesicular-arbuscular mycorrhiza on the growth of Griselinia littoralis (Cornaceae). Neiv PhytoL, 58, 274. BUTLER, E. J. (1939). The occurrences and systematic position of the vesicular-arbuscular type of mycorrhizal fungi. Trans. By. mvcol. Soc, 22, 274. CL.^RK, F. B. (1963). Endotrophic mycorrhizae mruence yellow poplar seedling growth. Science, 140,1220. D.\FT, M. J. (1965). Some interactions of kinetin and temperature on tohacco leaves infected with tomato aucuba mosaic virus. Ann. appl Biol., 55, 51. G.\DGIL, P. D. (1965). Distribution of fungi on living roots of certain Gramineae and the effect of root decomposition on soil structure. PI. Soil, 22, 239. GERDE.MANX, J. \V. (1955). Relation of a large soil-borne spore to phycomycetous mycorrhizal infections. Mycologia, 47, 619. GERDEM.-\NN, J. W. (1961). A species of Endogone from corn causing vesicular-arbuscular mycorrhiza. Mycologia, 53, 254. GERDEM.^XX, J. ^^ \ (1964). The effect of mycorrhiza on the growth of maize. ^Iycologia, 56, 342. GERDEM.AKN', J. W. (1965). ^'esicular-arbuscular mycorrhizae formed on maize and tulip tree by Endogone fasciculata. jmycologia, 57, 562. GERDEMANN, J. W. & NICOLSON, T. H. (1963). Spores of mycorrhizal Endogone species extracted from soil by wet sieving and decanting. Trans. Br. mycol. Soc, 46, 235. HARLEY, J. L. (1959). The Biology of Mycorrhiza. Leonard Hill, London. H.ARLEY, J. L. (1965). Mycorrhiza. Svmposiuni on Ecology of Soil-borne Plant Pathogens, p HEWITT, E. J. (1952). Sand and water culture methods used in the study of plant nutrition. C.A.B. Technical Cotnmiintcation, No. 22. HoLEV.AS, C. (1964). Private communication. University of Bristol, Long Ashton Research Station. JONES, F. R. (1924). A mycorrhizal fungus in the roots of legumes and some other plants, jf. agric. Res., 29, 459- KOCH, H. (1961). L'ntersuchungen liber die Mykorrhiza der KulturpHanzen unter besonderer Berucksichtigung \ on Althaea officinalis L., Atropa belladonna L., Helianthus anniius L. und Solanum lycopersicuiii L. Gartenbamvissenschaft, 26, 5. MELOH, K.-A. (1963). L^ntersuchungen zur Biologie der endotrophen Mycorrhiza bei Zea mays L. und Arena sativa L. Arch. Ahkrobiol., 46, 369. MossE, B. (1956). Fructifications of an Endogone sp. causing endotrophic mycorrhiza in fruit plants. Ann. Bot., 20, 349. MosSE, B. (1959). Observations on the extra-matrical mycelium of a vesicular-arbuscular endophyte. Trans. Br. mycol. Soc, 42, 439. MossE, B. (1963). Vesicular-arbuscular mycorrbiza: an extreme form of fungal adaption. Symp. Soc gen. ^licrobiol., 13, 146. NICOLSON, T. H. (i960). Mycorrhiza in the Gramineae. IL Development in different habitats, particularly sand dunes. Trans. Br. niycol. Soc, 43, 132. NICOLSON, T. H. & GERDEM.A,NN, J. W. (1962). Unpublished results: Life cycles of three Endogone mycorrhizal species. Department of Botany, Queen's College, Dundee. OTTO, G. (1962). Das Auftreten und die Entwicklung der endotrophen Mykorrhiza an ein- bis dreijahrigen Apfelsamlingen auf verschiedenen Standorten. L Teil: Haufigkeit, Intensitat und morphologische Merkmale der endotrophen Mykorrhiza. Zentbl. Bakt. ParasitKde, 115, 404; IL Teil: Beziehungen zwischen der endotrophen Mykorrhiza und den Faktoren Pflanz und Standort. Zentbl. Bakt. ParasitKde, 115, 525. OTTO, G. (1963). Uber die endotrophe Mykorrhiza der Obstgeholze und ihre Wechselbeziehungen zum Standort und zur Wirtspflanze. Internationales Mykorrluzasvniposium, Weimar, i960. STEVENSON, G. (1964). The growth of seedlings of some pioneer plants and the microorganisms associated with their roots. Trans. Br. mycol. Soc, 47, 331. THORNTON, H. G. ( ). Soil Microbiology Department of Rothamsted Experimental Station. Work of Dr. J. Mollison on mycorrhiza in wheat and clover. Rep. Rothamsted exp. Stn, 1950, p. 51; 1951, p. 58. WASTIE, R. L. (1965). The occurrence of an Endogone type of endotrophic mycorrhiza in Hevea brasiliensis Trans. Br. mycol. Soc, 48, 167. WILHELM, S. (1959). Parasitism and pathogenesis of root-disease fungi. Plant Pathology, pp University of Wisconsin Press, Madison. WINTER, A. G. & PEUSS-SCHONBECK, H. (1963). Zur Bedeutung der endotrophen Mykorrhiza fur die Entwicklung von KulturpHanzen. Internationales Mykorrhizasympositim, Weimar i960.

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