EFFECT OF ENDOGONE MYCORRHIZA ON PLANT GROWTH

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1 New PhytoL (1974) 73, EFFECT OF ENDOGONE MYCORRHIZA ON PLANT GROWTH VII. INFLUENCE OF INFECTION ON THE GROWTH AND NODULATION IN FNCH BEAN {PHASEOLUS VULGARIS) BY M. J. DAFT AND A. A. EL-GIAHMI Department of Biological Sciences, University of Dundee {Received 18 May 1974) SUMMARY Infections of Phaseolus with Endogone and Rhizobium compared with Rhizobium alone had increased growth, reproduction, number and weight of nodules, acetylene reduction rates, leghaemoglobin, phosphorus and total protein contents. Shoot/root ratios were lower in the dual infected plants. Levels of infection by Endogone influenced nodulation and the application of soluble phosphate simulated the effects of the fungus. The significance of these additive effects of dual symbiotic infections is discussed. INTRODUCTION Micro-organisms in the rhizosphere can increase or decrease the absorption of inorganic nutrients by plant roots. These effects depend on the element, its availability and the physical conditions around the roots (Barber, 1973). Phosphate has a profound influence on plant metabolism and growth and its economy in nature is of importance. Mycorrhizal plants are more efficient in the uptake of phosphate (Mosse, 1973) and in legumes, phosphate stimulates nodule production and the rate of fixation of atmospheric nitrogen (van Schreven, 1958). Some crop plants, such as legumes, are both mycorrhizal and nodulated. Asai (1944) indicated the influence of mycorrhizal infection on nodulation in legumes, but his work has only recently been fully appreciated. Nodulated soybeans grown in fumigated soil responded to inoculation with Endogone mosseae and their yield was increased by 29% (Ross and Harper, 197). Ross (1971) suggested that non-mycorrhizal soybean roots were inefficient phosphate-absorbing organs and that mycorrhizal infection promoted the uptake of phosphate. In this paper, we describe the effects of Endogone infection on various aspects of nodulation in French beans by comparing nodulated only with doubly infected, nodulated and mycorrhizal, plants. MATERIALS AND METHODS Growth and inoculation of plants. Seeds of French bean {Phaseolus vulgaris var. Canadian Wonder) were germinated in sterile sand. Seedlings, selected for uniformity, were transplanted singly into 4-in. pots containing sand plus bonemeal (level.5, Daft and Nicolson, 1966). At this time the roots were inoculated with a i ml suspension of 1139

2 114 M. J, DAFT AND A. A. EL-GIAHMI Rhizobium phaseoli (1* cells ml ^) and forty-five spores of Endogone macrocarpa var. geospora. In one experiment, five and fifty spores were used as the fungal inocula. In order to make the microflora more equivalent at the start of the experiments, the Rhizobium only controls received the same volume of the fungal inoculum which had been filtered three times to remove all Endogone spores and mycelium. The plants were grown under controlled environmental conditions of 13-h day length, a light intensity of 45 ft candles and at 25 C. Weekly, each plant received 25 ml of Long Ashton nutrient solution at half the recommended concentration. Unless stated, the phosphate and nitrate ions were substituted by other ions (Hewitt, 1952). Growth parameters measured. Growth of the plants was measured by the following: height, number of leaves, dry weight of shoots and roots, the numbers of flowers and fruits, and dry weights of fruits. Estimates of nodulation and mycorrhizal infection. The nodules from each plant were counted, weighed and their nitrogenase activity determined by the method of Sprent (1969). For each plant, the nodules were detached from the roots, washed, blotted dry and 1 mg were placed in 7-ml serum bottles. After replacing the air with a mixture of argon (77.9%), oxygen (22%) and carbon dioxide (.4%),.7 ml of acetylene was added to the bottles. Samples were taken after 45-6 min incubation at room temperature and analysed in a gas chromatogram for ethylene production. The leghacmoglobin content of the nodules was determined using the method of Keilin and Hartree (1951) and percentage mycorrhizal infection as by Daft and Nicolson (1972). Chemical analysis. Total protein and phosphorus contents of the shoots were analysed as described by Jackson (1958). Table i. Effect /Endogone and Rhizobium infections on the growth and reproduction of bean plants Days from inoculation with endophyte(s) 35 Rt 49 R 63 R Meant height (cm) ** ** 53.7** Mean no. of leaves ** 6.7** ** Mean no. of flower ** Mean no. of fruit ** ** * Mean dry weights of fruit (g) t Mean of eight plants. ** and * Significant at i and s% levels. t R = Inoculated with Rhizobium only. = Inoculated with Rhizobium and Endogone. Mean total dry weight of shoots (g) I.I ' 4 *-3 Mean % fungal infection of root _ 32 6 _ 63 SULTS In two similar experiments, all plants were inoculated with the Rhizobium suspension (R) and half were also given the Endogone inoculum (). Effect of Endogone on the growth of nodulated plants In experiment A, eight R and bean plants were harvested after 35, 49 and 63 days. There was only a small increase in plant height in the R treatment at the final harvest whereas, in the plants, maximum height was reached after 49 days (Table i). At all three harvests, the plants were significantly taller than their corresponding

3 Endogone mycorrhiza. VII 1141 control plants (R). Plate i. No. i shows representative plants from the R and treatments. plants possessed significantly more leaves at each harvest. They retained about the same number of leaves but, in the R treatment, the number of leaves decreased. Premature leaf fall is typical of non-mycorrhizal plants grown under conditions of poor nutrition and this has been shown for tomato (Daft and Nicolson, 1969b) and maize plants (Daft and Hacskaylo, 1974). By the first harvest the plants were in flower and the plants had produced three times the number of flowers on the control R plants. Flowering had finished by the second harvest. Although at each harvest the plants had developed significantly more fruits, the number varied from harvest to harvest because many did not develop fully and fell prematurely. The dry weight of the mature fruits was also greater from the plants. The R plants reached maximum weight after 49 days' growth and then declined due to excessive premature leaf and fruit fall. In plants, there was no difference between the last two harvests. The mycorrhizal infection approached and maintained its maximum level 49 days from inoculation s Days from inoculation Fig. I. Effect of mycorrhizal infection on the dry weights of shoots (O, nodulated and mycorrhizal; A, nodulated) and roots (, nodulated and mycorrhizal; A, nodulated) of Phaseolus vulgaris. 49 Experiment B investigated the effect of the single and double symbiotic associations in more detail. The growth of the shoots and roots in the plants was faster than in the R plants (Fig. i). In the shoots, maximum weight occurred 35 days from inoculation. Although more marked in the R plants, both treatments then showed a decrease in dry weight due to premature leaf and fruit drop. The roots in both treatments stopped growing after about 35 days from inoculation but neither root system decreased in size from their maxima to the end of the experiment. Throughout the experiment the shoot/root ratios from the plants were always lower than R plants. In both, the ratios decreased to the third harvest (28 days), reached a maximum 35 days from inoculation and then decreased again.

4 1142 M, J. DAFT AND A, A, EL-GIAHMI Effect of Endogone on nodule development At each harvest in experiment A, plants produced significantly more nodules than R plants. (P<o.oi, Table 2). The maximum number of nodules was found after 49 days' growth in the treatment, whereas, in the R treatment, there was an increase from the first to the last harvest. Nodules from the plants weighed significantly more than those from the R plants 35 and 49 days from inoculation (P<o.oi) but there was no difference after 63 days when the nodules were beginning to senesce. At each harvest, the rate of acetylene reduction per mg nodule (Table 2) was greater in the nodules from the plants with double infection () than in those from the R treatment, although only at the second harvest was the difference statistically significant. However, as the plants possessed a greater weight of nodular tissue, the difference per plant between the two treatments was much greater. That this is not just a function of the larger size of the plants was confirmed in a similar experiment not reported here in which the ratio of dry weight of nodules/total dry weight of plant was.65 +o-ooo6 for the R treatment and.15 ±.2 for the treatment and the difference was significant (P<o.oi) showing that the roots produced a greater weight of nodular tissue per unit of root than the R Table 2, Effect of Endogone infection on nodulation and acetylene reduction in bean plants Days from inoculation with the endophyte 35 Rt 49 R 63 R Meant no. of nodules/plant ** 98.3 i68.6** ios.o 126.** Mean fresh wt of nodules (g)/plant.41.58**.48 o.86*» Mean rate of acetylene reduction p moles mg fresh wt nodule 19 J3 aa* aa 2g % Mean of eight replicates. ** and * Significant at the 1% and 5% level respectively. t R and as in Table i. CjHi/min/ Total fresh wt nodules/plant alone. As acetylene reduction is an indirect measurement of the nodules' ability to fix atmospheric nitrogen, it follows that -infected plants are more efiscient in the fixation of atmospheric nitrogen and the absorption of phosphorus. Analyses of the shoot tissues gave 19 and 25% total protein content and 1275 ^nd 2925 ppm of phosphorus in the R and plants respectively. In experiment B, three aspects of nodule development were investigated along with the development of the mycorrhizal infection. Fig. 2 records weekly samples of the concentration of leghaemoglobin in the nodules and their number and weight. Leghaemoglobin concentration (Fig. 2a) remained higher in the treatment throughout the experiment, even during the decline period (35-56 days). The number and weight of nodules from the plants were greater than from the R plants (Fig. 2b, c). Maximum numbers of nodules were produced in both treatments after 35 days, whereas maximum fresh weight was earlier in the treatments (28 days) than in the R treatment (after 35 days). The French bean plants respond very quickly to infection with Endogone because after 21 days' growth the infection level was only c. 2% (Fig. 2d). The mycorrhizal development increased gradually to a maximum of 57% after 56 days.

5 Endogone mycorrhiza. VII (c) Days from inoculation Fig. 2. EfFect of mycorrhizal infection on (a) leghaemoglobin content of the nodules; (b) number of nodules produced; (c) fresh weight of the nodules and (d) the development of the mycorrhizal infection (, nodulated and mycorrhizal; A, nodulated) in beans. Table 3. Effect of two concentrations of Endogone spores on the growth, nodulation and acetylene reduction in bean plants grown for 49 days Meant dry weight (g) Shoot Root Mean shoot/root ratio Mean no. nodules/plant Mean fresh weight nodules (R)/plant Mean rate of acetylene reduction p moles C2H4/min/ mg fresh wt nodule Total fresh wt nodules/plant Concentration of Endogone spores in fungal inoculum Control () I.IO ' Low (5) ' ^ I.SO High (5) o.« ± ± ± ±.21 I-37 ± t Mean of sixteen plants. 3a 35493

6 II44 M. J. DAFT AND A. A, EL-GIAHMI Effect of the concentration of the Endogone inoculum on Rhizobium infected bean plants. As spore concentration in the inoculum and the resultant level of infection within the root system has been shown to affect the growth of tomato plants (Daft and Nicolson, 1969b) the influence of Endogone inoculum on bean plants was investigated by using two concentrations of spores (five and fifty per plant). The plants in experiment C were harvested after 49 days. The dry weight of both roots and shoots were highest with the higher concentration of Endogone spores (Table 3). Both mycorrhizal treatments gave lower shoot/root ratios than the control treatment (nodulated alone). Nodule numbers per plant, their weight and efficiency of acetylene reduction were least in the Rhizobium alone treatment and greatest in the nodulated and high Endogone spore treatment. Nodules taken from representative plants from each treatment are shown in Plate i, No. 2. The mycorrhizal infections for the five spores and fifty spores/ plant treatments were 27 and 46% respectively. Expressing the fresh weight of^ nodules per g dry weight of the whole plant showed that a statistically significant (P<o.O5) greater mass of nodular material was formed on the two treatments. The mean ratio for the R plants was.49 and.61 and.6 for the low and high Endogone spore concentrations respectively. These results show that the level of the Endogone inoculum and hence the degree to which the mycorrhiza develops affect the growth and nodulation of the host. Table 4. Effect of mycorrhizal infection on the production of nodules and their rate of acetylene reduction in bean plants grown for 49 days and given different N and P treatments Treatment (-N-P) R(-N"P) R(-N+P) R(+N4P) Meant no. of nodules io4±7.i ±5. Mean fresh weight of nodules (mg) O±2.o Mean rate of acetylene reduction p moles C2H4/ mg fresh wt 28±3.6 i6±2.i 22±3.8 9±i.8 t Mean of nine replicates. R, Rhizobium. E, Endogone. N, Nitrate. P, Phosphate. Effect of soluble phosphate and nitrate on Rhizobium and Endogone infected bean plants. Experiment D in this investigation shows the infiuence of soluble phosphate and nitrate on Rhizobium and Endogone infected beans. All the plants were grown in sand containing bonemeal (level.5) and, in each of the following treatments, there were nine replicates, (i) Plants infected with Rhizobium and Endogone and fed with nutrient solution minus both nitrate and phosphate ions, (-N-P). (ii) Plants infected with Rhtzobium and fed with nutrient solution minus both nitrate and phosphate ions, R (-N-P). (iii) Plants infected with Rhizobium and fed with nutrient solution minus the nitrate ion, R (-N +P). (iv) Plants infected with Rhizobium and fed with the complete nutrient solution, R ( 4-N +P). Nodule numbers were highest in the ( N-P) treatment, lowest in the treatments, R (-N-P) and R ( +N +P), and intermediate in treatment R (-N +P) (Table 4). The difference between the two extreme treatments was significant at the 5% level of probability i.e. (-N-P)>R (-N-P) and R (+N+P). Nodule weights showed

7 Endogone mycorrhiza. VII 1145 similar diflferences amongst the treatments. Treatments ( N P) and R ( N +P) gave a greater vi^eight of nodules than each of the R (-N -P) and R ( +N +P) treatments, and the differences were significant (P<o.o5). The acetylene reduction rates were highest in the (-N -P) treatment followed by R (-N +P), R (-N-P) and finally R ( +N +P). These results show that nodule production and nitrogenase activity were maximum when the plants were given phosphate but no nitrate and that this phosphate can be applied in solution or presumably supplied via the mycorrhiza. DISCUSSION This investigation supports the work of Asai (1944) and Ross and Harper (197) that the two major elements, nitrogen and phosphorus, can be supplied to the host plant by means of symbiotic associations. Elements such as phosphorus (van Schreven, 1958), zinc (Demeterio, Ellis and Paulsen, 1972) and copper (Hewitt, 1958) influence nodulation and nitrogen fixation in higher plants. A greater concentration of these elements has been shown to occur in mycorrhizal plants. Mycorrhizal peach trees contained more zinc than non-mycorrhizal trees which showed deficiency symptoms (Gilmore, 1971). In soybeans and alfalfa with dual infections, the concentrations of copper and phosphorus were higher (Ross and Harper, 197; Daft and Hacskaylo, 1974). It is not known if the enhanced uptake of nutrients by the mycorrhiza directly affects the bacteroids, if the bacteroids affect the mycorrhiza or if they both interact by means of the nutrition of the host. The leghaemoglobin content of the bacteroids and nitrogen fixation have been shown to be related (Virtanen et al., 1947) and more recently Bergersen (1971) has reviewed the proposed physiological roles of leghaemoglobin in nitrogen fixation. Further experiments (Bergersen and Goodchild, 1973) support the suggested role of leghaemoglobin in stimulating oxygen uptake and nitrogenase activity by the bacteroids. The parameters measured in this investigation i.e. plant size, weight, numbers, relative amount of tissue, concentration of leghaemoglobin of nodules and rates of acetylene reduction were all higher in the mycorrhizal plants. The intensity of the mycorrhizal infection positively influenced these parameters. Because the shoot/root ratio was always less in the treatment, it suggests that the mycorrhizal infection produces a more actively growing root system although the ratios vary depending on the stage of development of the host. The effects of mycorrhizal infection discussed in this paper can be replaced by the addition of soluble phosphate. Soluble phosphate increases nitrogenase activity in bluegreen algae (Stewart and Alexander, 1971) and, in arctic tundra, it has been suggested as a major factor affecting the rates of atmospheric nitrogen fixation (Schell and Alexander, 1973)- Daft and Nicolson (1969a) showed that the addition of soluble phosphate depresses the development of the mycorrhizal infection in maize. Furthermore, in three membered systems, virus production was greater in mycorrhizal hosts (Daft and Okusanya, 1973)- It was suggested that this increased virus production was due to the enhanced uptake of phosphate by the mycorrhizal plants. Non-mycorrhizal tomato plants grown under the same conditions, infected with tomato aucuba mosaic virus and given soluble phosphate gave similar yields of virus. In areas throughout the world deficient in phosphorus, application of phosphatic fertilizers may also lead to an increase in the nitrogen content of the soil. Tropical legumes (Andrew and Robins, 1969) and subterranean clover in Australia (Watson, 1969) and in New Zealand (Jones, Oh and Ruckman, 197) responded to the addition of

8 1146 M. J, DAFT AND A, A. EL-GIAHMI soluble phosphorus. The yields of grain legumes in India (Prased, Bhendia and Bains, 1963) and of Glycine javanica in Brazil (Souto and Dobereiner, 1968) were greater after fertilizing with phosphorus. Harley (197) has written that plants with dual symbiotic associations are particularly successful as primary colonizers owing to their ability to compensate for the infertility of the habitat. On the coal wastes of Pennsylvania, Daft and Hacskaylo (1974) noted the success of plants such as Robinia, Coranilla and Eleagnus, all nodulated and mycorrhizal. The growth of nodulated plants on poor agricultural soils is a common practice. Great care is taken in selecting the most efficient strains of bacteria and our results suggest that the fungal endophyte also needs careful consideration. ACKNOWLEDGMENTS We thank the Libyan Government for a Research Studentship to one of us (A.A.E,) and Drs J. I. Sprent and T. H. Nicolson of this department for helpful discussions and continued interest, FENCES ANDW, C. S. & ROBINS, M. F. (1969). Effect of phosphorus on the growth and chemical composition of some tropical pasture legumes. II. Nitrogen, calcium, magnesium, potassium and sodium contents. Aust.y. agric. Res., ao, 675. AsAi, T. (1944). (Jber die Mykorrhizenbildung der Leguminosen Pflanzen. jfap. jf. Bot., 13, 463. BARBER, D. A. (1973). Effects of micro-organisms on the absorption of inorganic nutrients by plants. Pestic. Sci., 4, 367. BERGERSEN, F. J. (1971). Biochemistry of symbiotic nitrogen fixation. A. Rev. PI. PhysioL, aa, iai. BERGERSEN, F. J. & GOODCHILD, D. J. (1973). Cellular location and concentration of leghaemoglobin in soybean root nodules. Aust. J. hiol. Sci., a6, 741. DAFT, M. J. & HACSKAYLO, E. (I975)- Vesicular-arbuscular mycorrhizas in the anthracite and bituminous coal wastes of Pennsylvania (in preparation). DAFT, M. J. & NICOLSON, T. H. (1966). Effect of Endogone mycorrhiza on plant growth. New Phytol., 65, DAFT, M. J. & NICOLSON, T. H. (1969a). Effect of Endogone mycorrhiza on plant growth. II. Influence of soluble phosphate on endophyte and host in maize. Nezii Phytol., 68, 945. DAFT, M. J. & NICOLSON, T. H. (1969b). Effect of Endogone mycorrhiza on plant growth. III. Influence of inoculum concentration on growth and infection in tomato. New Phytol., 68, 953. DAFT, M. J. & NICOLSON, T. H. (1972). Effect of Endogone mycorrhiza on plant growth. IV. Quantitative relationships between the growth of the host and the development of the endophyte in tomato and maize. New Phytol., 71, 287. DAFT, M. J. & OKUSANVA, B. O. (1973)- Effect of Endogone mycorrhiza on plant growth. V. Influence of infection on the multiplication of viruses in tomato, petunia and strawberry. New Phytol., 7a, 975. DEMETERIO, J. L., ELLIS, R. & PAULSEN, G. M. (1972). Nodulation and nitrogen fixation by two soybean varieties as affected by phosphorus and zinc nutrition. Agron.J., 64, 566. GiLMO, A. E. (1971). The influence of endotrophic mycorrhizae on the' growth of peach seedlings. J. Amer. Soc. Hort. Sci., 96, 35. HARLEY, J. L. (197). The importance of micro-organisms to colonizing plants. Tram. Bot. Soc. Edinb., 41, 65- HEWITT, E. J. (1952). Sand and water culture methods used in the study of plant nutrition. C.A.B. Technical Communication, No. 22. HEWITT, E. J. (1958). Some aspects of mineral nutrition in legumes. In: Nutrition of the legumes (Ed. by E. G. Hallsworth), p. 15. Butterworths, London. JACK.SON, M. J. (1958). Soil Chemical Analysis. Constable & Co. Ltd., London. JONES, M. B., OH, '}. H. & RUCKMAN, J. E. (197). Effect of phosphorus and sulphur fertilization on the nutritive value of subterranean clover (Trifolium subterraneum). Proceedings of the New Zealand Grassland Association, 3a, 69. California University, Hopland, U.S.A. KEILIN, D. & HARTE, E. F. (19.S1). Purification of horse radish peroxidase and comparison of its properties with those of catalase and methaemoglobin. Biochem. J. 49, 88. Mos.SE, B. (1973). Advances in the study of vesicular-arbuscular mycorrhiza. Ann. Rev. Phytopath., 11, 171. PRASAD, R., BHENDIA, M. L. & BAINS, S. S. (1968). Re.sponse of grain legumes to levels and sources of phosphorus on different soils. Indian J. Agron., 13, 35. Ross, J. P. & HARPER, J. A. (197). Effect of Endogone mycorrhiza on soybean yields. Phytopathology, 6, Ross, J. P. (1971). Effect of phosphate fertilization on yield of mycorrhizal and non-mycorrhizal soybeans. Phytopathology, 61, 14.

9 THE NEW PHYTOLOGIST, 73, 6 PLATE I M. J. DAFT AND A. A. I'L-CHAHMI ENDOGONK MYCORRHIZA. VII (f,,cing p. 1146)

10 Endogone mycorrhiza. VII ScHELL, D. H. & ALEXANDER, V. (1973). Nitrogen fixation in arctic coastal tundra in relation to vegetation and micro-relief. Arctic, a6, 13. SCHVEN, D. A. VAN (1958). Some factors affecting the uptake of nitrogen by legumes. In: Nutrition of the Legumes. (Ed. by E. G. Hallsworth), p Butterworths, London. SouTO, S. M. & DoBEiNER, J. (1968). Effect of phosphorus, soil temperature, soil moisture on nodulation and growth in two varieties of Glycine javanica L. Pesq. agropec. bras., 3, 215. SPNT, J. I. (1969). Prolonged reduction of acetylene by detached soybean nodules. Planta (,Berl.),SS, 372. STEWART, W. D. P. & ALKXANDER, G. (1971). Phosphorus availability and nitrogenase activity in aquatic blue-green algae. Freshwat. BioL, 1, 389. ViRTANEN, A. I., JoRMA, J., LiNKOLA, H. & LiNNASALMi, A. (1947). On the relation between nitrogen fixation and leghaemoglobin content of leguminous root nodules. Acta Chem. Scand., I, 9. WATSON, E. R. (1969). The influence of subterranean clover pastures on soil fertility. III. The effect of applied phosphorus and sulphur. Aust. Jf. agric. Res., ao, 447. EXPLANATION OF PLATE PLATE 1 No. I. Bean plants grown for 63 days and infected with Rhizobium (R) and Rhizobium plus Endogone (). No. 2. Nodules from bean plants infected with Rhizobium (Ri) and Rhizobium plus Endogone ( ii and iii), five and fifty spores/plant respectively. No. 3. Root systems from bean plants infected with Rhizobium (R) and Rhizobium plus Endogone ().

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