INTERACTION BETWEEN A VESICULAR-ARBUSCULAR MYCORRHIZA AND RHIZOBIUM AND THEIR EFFECTS ON SOYBEAN IN THE FIELD

New Phytol. (1979) 82. 141-145 I j_i INTERACTION BETWEEN A VESICULAR-ARBUSCULAR MYCORRHIZA AND RHIZOBIUM AND THEIR EFFECTS ON SOYBEAN IN THE FIELD BY D. J- BAGYARAJ, A. MANJUNATH AND R.B. PATIL Department of Agricultural Microbiology, University of Agricultural Sciences, Bangalore-560065y India {Accepted 3 May 1978) SUMMARY Interaction between fasciailatiis and Rhizobium japonicum and their effects on soybean in the field was studied in a phosphorus deficient sandy loam soil with ph 5 6. The number, dry weight and nitrogen content of the root nodules in plants inoculated with plus RJiizobium were significantly more compared to plants inoculated with only Rhizobium. Rhizobium inoculation did not have any significant eitect on sporulation of CJ. fasciculatus in the rhizosphere. Although soybean plants inoculated witb G. fasciculatus recorded increased phosphorus content, dry weight and grain yield than uninoculated plants the differences were not statistically significant. In RJiizobium only inoculation markedly increased the nitrogen content of the plant and grain yield. Dual inoculation with both the symbionts increased significantly the dry weight of the shoot and its nitrogen content over single inoculation with either or RJiizobium. These results suggest that vesicular-arbuscular (VA) mycorrliiza can greatly assist nodulation and nitrogen fixation in field growia soybean inoculated with rhizobia. INTRODUCTION Research in the last few decades has established that vesicular-arbuscular (VA) mycorrhiza can improve plant growth through increased uptake of phosphorus, especially in soils of low fertility (Gerdemann, 1975). Asai (1944) demonstrated that several legumes grow poorly and failed to nodulate in autoclaved soil unless they were mycorrhizal. This was probably due to phosphorus deficiency since an adequate phosphorus supply is important for satisfactory nodulation and nitrogen fixation (Demeterio, Ellis and Paulsen, 1972). Ross and Harper (1970) showed that the growth and the yield of nodulating soybean increased after inoculation with mosseae in fumigated soil. Inoculation of crop plants with VA mycorrhizal fungi and Rhizobium was found to have synergistic beneficial effect on nodulation, nitrogen fixation and plant growth (Daft and El-Giahmi, 1976; Mosse, 1977a, b). These studies were carried out in pots or bins using sterilized or unsterile soil or fumigated field soil. Response of soybean to dual inoculation with VA mycorrhiza and Rhizobium and the effect of each organism on the other in soybean rhizosphere, studied under natural field conditions are presented in this paper. 0028-646X/79/010i-0141 $02.00/0 ^6 1979 The New Phytologist

142 D. J. BAGYARAJ ETAL. MATERIALS AND METHODS The experiment was conducted in a non-cultivated fallow covered with graminaceous weeds, mainly Cynodon dactylon (L.) Pers. The soil was sandy loam with ph 5-6, phosphorus-deficient (2-4 p.p.m., available P extracted with NH4F and HCL) and practically devoid of rhizobia nodulating soybean, but had an indigenous population of (198 per 50 ml soil) mycorrhizal spores. Urea, superphosphate and muriate of potash at the rate of 5 kg N, 5 kg P^Og and 5 kg KoO per ha respectively (i.e. half the recommended rate) were broadcasted before sowing. The experimental plots were 1-2 X 1-0 m and sown with soybean seeds [Glycine max (L.) Merr. cv. Hardee], giving 30 and 10 cm spacing between rows and plant respectively. There were four inoculation treatments: (1) uninoculated control, (2) inoculated with Rhizobium japonicum (Kirchner) Buchanan, (3) inoculated with the VA mycorrhizal fungus fasciculatus (Thaxt.) Gerd. and Trappe, and (4) inoculated with (2) and (3). Rhizobium inoculation was done by treating the soybean seeds with a peat based culture before sowing. Mycorrhizal inoculation was done by placing the seeds over a thin layer of the mycorrhizal inoculum at the time of sowing. The mycorrhizal inoculum consisted of roots and soil from a pot culture of Sudangrass [Sorghum bicolor (L.) Moench var. sudanense] which was infected with fasciculatus and grown for 4 months. The inoculum contained hyphae, vesicles, Chlamydospores and arbuscules of G. fasciculatus. There were six replications for each treatment. Since the experiment was conducted during monsoon, only protective irrigation was given twice to save the crop. At two intervals, i.e. 45 and 60 days after sowing, observations on the number and dry weight of nodules and dry weights of root and shoot were taken. Phosphorus content of the shoot was determined colorimetrically by the Vanadomolybdate/ phosphoric-yellow colour method outlined by Jackson (1967). Total nitrogen determinations were made by the microkjeldahl method (Bremner, 1960). Percentage mycorrhizal infection of the root was determined by the root slide technique (Nicolson, 1960) after clearing the roots with KOH and staining with trj^pan blue (Phillips and Hayman, 1970). Numbers of G. fasciculatus spores in the soil surrounding the roots were determined by the wet sieving and decanting technique (Gerdemann and Nicolson, 1963). Grain yield and 100-grainweightwererecordedat the time of harvest. RESULTS The number, dry weight and nitrogen content of the soybean root nodules as influenced by Rhizobium and mycorrhizal inoculation is given in Table 1. There was no nodulation at all in the uninoculated control plants and in the plants inoculated only with the mycorrhiza. Plants treated with Rhizobium nodulated well. The number, size, dry weight and nitrogen content of nodules in plants inoculated with mycorrhiza plus Rhizobium were significantly greater than those of plants inoculated with Rhizobium only, 60 days after inoculation but not at 45 days after inoculation. Plants inoculated with G. fasciculatus showed a significantly greater mycorrhizal spore count in the rhizosphere compared to the uninoculated control plants and plants which received only Rliizobium (Table 2). These increases were more striking when the plants were 60 days old than 45 days. The percentage mycorrhizal infection of the roots was also greater in plants inoculated with G. fasciculatus.

VA mycorrhiza and Rhizobium in soybean 143 The dry weight of plant tops, grain yield and 100-grain weight are presented in Table 3. The differences in the dry weight of the plant tops were not significant in 45-day-oId plants. Dual inoculation with and Rhizobium produced significantly larger plants compared to uninoculated control plants and plants inoculated with either or Rhizobium. The grain yield of plants treated with Rhizobium alone or Rhizobimn plus was significantly greater than that of uninoculated plants Table 1. Number, dry weight and nitrogen content of the soybean root nodules as influenced by inoculation with fasciculatus and Rhizobium japonicum Treatment RJiizobium + RJiizobium Nodule number per 20 b 31b plant 25 b 37c Nodule dry wt per (ff) ; ' 0 07 b 0-12b plant i 024b 0 4ic IN'odule nitrogen per plant (mg) 2 52b 329b S69b 17-23c 45 = 45 days after sowing; 60 60 days after sowing. * Significant at P =^ 0 05. Values not followed by identical letters in each vertical column are significantly different. Analysis done after ^,'(-v-^0 05) transformation of the actual values. Table 2. Mycorrhizal spores in soil and % mycorrhizal infection of soybean roots as influenced by inoculation with fasciculatus and Rhizobium japonicum Spore count per 50 mi soil O-" infectionf Treatment 45 60 RJiizobium -\-RJiizobium 226 a 260 ab 226a 274 b 227 a 268 b 225a 290 b 60 69 65 82 75 88 86 93 45 = 45 days after sowing; 60 = 60 days after sowing. * Significant at P = 0-05. Values not followed by identical letters in each vertical eolumn are significantly different. t Percentage infection values did not lend themselves to statistical analysis. Table 3. Shoot dry weight, grain yield and 100-grain weight of soybean as influenced bv inoculation with fasciculatus and Rhizobium japonicum Treatment Rhizobium + RJiizobium Shoot 43NS 098 1-25 1-41 M9 dry wt (g) per plant l%a 2-59a 283 a 4 65 b Grain yield per plot (g) 44-49 a 56-25 a 84-98 b 100-71 b 100-grain wt 18) 1078a 10S7a 14-55b 1464b 45 = 45 days after sowing; 60 = 60 days after sowing. NS = Not significant. * Significant at P = 0 05. Values not followed by identical letters in each vertical column are significantly different.

144 D. J. BAGYARAJ ETAL. and plants inoculated with only. The 100-grain weight of soybean seeds was significantly greater in plants inoculated with Rhizobium with or without. The nitrogen and phosphorus content of the shoot at two different intervals are presented in Table 4. No significant differences could be seen in 45-day-oId plants. In 60-day-old plants the nitrogen and phosphorus content of the plants which received plus Rhizobium were significantly higher compared to the uninoculated control plants, and plants which received only or Rhizobium. Table 4. Nitrogen atid phosphorus content of the soybean shoot as influenced by inoculation with fasciculatus and Rhizobium japonicum Total nitrogen content per shoot (mg) Total phosphorus content per shoot (mg) Treatment 45 N'8 60' RJiizobium + Rhizobium 33-83 37-55 39-09 4722 48-39a 72-74a 93-40ab 198-llc 3-64 5-24 4-82 503 U-89a 14-89a ll-26a 26-64b 45 = 45 days after sowing; 60 = 60 days after sowing. NS = Not significant. * Significant at P = 0-05. Values not followed by identical letters in each vertical column are not significantly different. DISCUSSION Improvement by inoculation with VA endophytes of the phosphate supply to the host plant and its growth and yield under natural field conditions in soils low in phosphorus has been obtained earlier in corn and wheat (Khan, 1975) and in potatoes (Black and Tinker, 1977). But Jackson, Franklin and Miller (1972) found that there was no effect of mycorrhizal inoculation on soybean yield under natural field conditions where inoculation improved the growth of corn. In the present study even though inoculation with G. fasciculatus resulted m 25% increase in the total phosphorus content of the shoot, 32% increase in the shoot dry weight and 25% increase in the grain yield over the uninoculated control plants, the increases were not statistically significant. This is perhaps due to the presence of considerable quantities of indigenous endophytes in soil. There is also growing evidence that certain endophytes form preferential associations with certain host plants (Mosse, 1977b). Schenck, Kinloch and Dickson, (1975) conducting pot experiments using unsterile soil, observed that the response of different cultivars of soybean differed with the different mycorrhizal species. In view of this it may be that G. fasciculatus used in the present study is not very effective with 'Hardee' soybean when grown in the soil and the environment under study. The better response of soybean to Rhizobium inoculation could be attributed to the complete absence of soybean specific native rhizobia in the soil. The better plant growth in 60 days due to Rhizobium-only inoculation compared to control plants was not statistically significant, while grain yield at the time of harvest (90 days) was significantly greater, suggesting that the crop derived more benefit from fixed atmospheric nitrogen in later stages of plant growth. Hardy et al. (1968) pointed out that

VA mvcorrhiza and Rhizobium in soybean the nodule activity of soybean reached the maximum during pod filling and did not decline until after pod filling was complete. The present investigation brings out clearly that VA mycorrhiza can greatly assist nodulation and nitrogen fixation in field grown soybean plants inoculated with rhizobia. Similar results in pot experiments using unsterilized soil were obtained in Stylosanthes (Mosse, 1977a). Ross and Harper (1970) recorded 29% increase yield in soybean grown in fumigated field plots, due to double inoculation with endomycorrhiza plus Rhizobium over single inoculation with only Rhizobium. While the principal effect of mycorrhiza on nodulation is undoubtedly phosphate mediated, mycorrhiza may have other secondary effects, possibly of hormonal nature (Mosse, 1977b). It is a common practice to grow nodulated plants on poor agricultural soils to increase their fertility and effective strains of rhizobia are often used for treating seeds. The present study brings out that an effective VA mycorrhizal fungus could contribute to the efficiency of such a system, especially in phosphorus-deficient soils, even though native endophytes may be present. REFERENCES ASAI, T, (1944). Uber die Mykorrhizenbildung der Leguminosen Vi\Anzen. Japanese Journal of Botauv, 13, 463. BLACK, R. L. B. & TINKER, P. B. (1977). Interaction between effects of vesicular-arbuscular mycorrhiza and fertilizer phosphorus on yields of potatoes in the field. N^ature, 267, 510. BREMNER, J. M. (1960). Determinations of nitrogen in soil by Kjeldahl method. Journal of Agricultural Science, 55, 11. DAFT, M. J. & EL-GIAHMI, A. A. (1976). Studies on nodulated and mycorrhizal peanuts. Annals of applied Biology, 83, 273. DEMETERIO, J. L., ELLIS, R. JR & PAULSEN, G. M. (1972). Nodulation and nitrogen fixation by two soybean varieties as affected by phosphorus and zinc nutrition. Agronomy Journal, 64, 566. GERDEMANN, J. W. (1975). Vesicular-arbuscular mycorrhizae In: Development and Function of Roots (Ed. by J. G. Torrey & D. T. Clarkson), pp. 575-591. Academic Press, London, New York. GERDEMANN, J. W. & NICOLSON, T. H. (1963). Spores of mycorrhizal Endogone species extracted from soil by wet sieving and decanting. Transactions of the British Mycological Society, 46, 235. HARDY, R. W. F., HOLSTEN, R. D., JACKSON, E. K. & BURNS, R. C. (1968). The acetylene-ethylene assay for Ng fixation: laboratory' and field evaluation. Plant Physiology, 43, 1185. JACKSON, M. L. (1967). Soil Chemical Analysis. Prentiee Hall, New Delhi. JACKSON, N. E., ERANKLIN, R. E. & MILLER, R. H. (1972). Effects of vesicular-arbuseular mycorrhizae on growth and phosphorus content of three agronomic crops. Proceedings of the Soil Science Society of America, 36, 64. KHAN, A. G. (1975). Growth effects of VA mycorrhiza on crops in the field. In: Endomycorrhizas (Ed. hy F. E. Sanders, B. Mosse & P. B. Tinker), pp. 419 435. Academic Press, London, New York. NICOLSON, T. H. (1960). Mycorrhiza in the Gramineae. II. Development in different habitats, particularly sand dunes. Transactions of the British Mycological Society, 43, 132. MOSSE, B. (1977a). Plant growth responses to vesicular-arbuscular mycorrhiza. X. Responses of Stylosanthes and mm'ze to inoculation in unsterile soils. Netv Phytologist, 78, 277. MOSSE, B. (1977b). The role of mycorrhiza in legume nutrition on marginal soils. In: Exploiting the Legume-RJiizobium Symbiosis in Tropical Agriculture (Ed. by J. M. Vineent, A. S. Whitney & J. Bose), pp. 275-292. College of Tropical Agriculture, University of Hawaii, U.S.A., Miscellaneous Publications, no. 145. PHILLIPS, J. M. & HAYMAN, D. S. (1970). Improved procedures for clearing roots and staining parasitic and vesicular-arbuscular mycorrhizal fungi for rapid assessment for infection. Transactions of the British Mycological Society, 55, 158. Ross, J. P. & HARPER, J. A. (1970). Effect of Endogone mycorrhiza on soybean yields. Phytopathohgv 60, 1552. SCHENCK, N. C, KINLOCH, R. A. & DICKSON, D. W. (1975). Interaction of endomycorrhizal fungi and root-knot nematode on soybean In: Endomycorrhizas (Ed. by F. E. Sunders, B. Mosse & P. B. Tinker), pp. 607-617. Academic Press, London, New York.