Gnzman-Plazola. R.A.. R. Ferrera-Cerrato and JJX Etchevers. Centro de Edafologia, Colegio de Postgraduados, Montecillo, Mexico. LEUCAENA LEUCOCEPHALA, A PLANT OF HIGH MYCORRHIZAL DEPENDENCE IN ACID SOILS In order to obtain significant growth and fixation of atmospheric nitrogen in Leucaena leucocephala in acid soils it is necessary to use high rates of phosphate fertilizer (Moreno, 1981; Guzman-Plazola et al., 1983). One possible, less-expensive alternative is to inoculate leucaena with vesicular-arbuscular mycorrhizal (VAM) fungi, which can improve phosphorus uptake, particularly when this nutrient is found in small amounts or is barely assimilable (Munns and Mosse, 1980; Lara-Fernandez y Ferrera-Cerrato, 1986; Gardezi et al., 1988). It would be most beneficial to combine the benefits of mycorrhizal and Rhkobium symbiosis. In this work we have assessed the response of Leucaena leucocephala to Rhkobium and VAM inoculation. Our main purpose was to estimate the potential of this double inoculation to substitute for nitrogen and phosphate fertilizers in acid soils. Materials and Methods: An experiment was carried out under greenhouse conditions in a complete randomized block design. The principal effects and interactions of the following factors were evaluated: nitrogen (0 ppm N, 20 ppm N and Rhkobium loti), mycorrhiza (Glomus intraradices, and no inoculation), phosphorus source (phosphate rock and ordinary superphosphate) and P rate (0,50,100 and 150 ppm). The experiment consisted of 48 treatments, each repeated three times, with a duration of 130 days. Plastic pots were used as experimental units and were filled with 4 kg of soil dried in the open air. This soil was sandy-loam in texture, with 8.7 ppm P (Bray-1), 0.05 % N, ph = 5.3, and had been previously fumigated with methyl-bromide. Two seedlings of Leucaena leucocephala cv Peruana were transplanted to each pot. In each pot, as mycorrhizal inoculant, 50 g of soil with spores of Glomus intraradices plus 1 g of corn roots with 80% of colonization were applied. The units inoculated with R. loti received one milliliter of a broth that had approximately 10 rhizobia of the strain CIAT-1920, previously mixed with 1 g of CaCO/25 ml of broth. In the plants that were not inoculated a solution of CaCO of the same concentration was applied. Ammonium nitrate and phosphate rock from San Luis Potosi (15.3% P) or ordinary superphosphate were used as N and P sources. Irrigations were carried out with distilled water. A 150 ml of Long Ashton nutrient solution with no nitrogen nor phosphorus were applied every two weeks. The following variables were evaluated: (a) dry weight of aerial part, (b) diy weight of root, (c) height of plant, (d) dry weight of nodules, (e) nitrogen content in the aerial part, (f) phosphorus content in the aerial part and (g) mycorrhizal colonization. Results Growth and Nodulation. Inoculation of Glomus intraradices produced substantial increases in the leucaena growth. When the phosphorus rate was zero, the fungus induced increases over the control of 380, 2,280,1,790 and 460% in the height, above ground dry weights, and root and nodules of the plants, respectively (Figure 1). These effects were equivalent to application of 150 ppm P. Plants that did not receive mycorrhizal treatment showed a strong response to phosphate fertilizer. In general this effect was even more pronounced with ordinary superphosphate than with phosphate rock, but the differences, except the dry weight of nodules, were only significant in the treatment with 100 ppm P. Mycorrhizal plants presented a small response to phosphate fertilizer application, with the exception of the root dry weight in the
Figure 1. Effect of the interaction of Mycorrhiza x Rate of P2 O5 x P source over (a) height of-l. leucocephala, (b) diy weight of above ground biomass, (c) diy weight of roots, (d) dry weight of nodules. (E) Effect of the interaction of Mycorrhiza x N source on the dry weight of above ground biomass. +M = G lomus intraradices; -M = without endomycorrhiza. OS = ordinary superphosphate; PR = phosphate rock. 150 ppm P treatment applied as ordinary superphosphate. None of the other rates or sources of this element produced major effects other than those obtained with the mycorrhizal fungus in the absence of fertilization. Rhizobium loti produced significant effects only in plants inoculated with Mycorrhiza. The above ground dry weight in the plants inoculated with Rhizobium was higher than that of those inoculated only with the mycorrhizal fungus (Figure le). The application of 20 ppm N in all cases did not affect the behavior of these variables. Phosphorus and nitrogen content in above-ground biomass: Inoculation with G. intraradices produced increases over the control of 3,500 and 850% in the phosphorus and nitrogen content of the above ground biomass, respectively (Figure 2a and b). The effect of this fungus on the first variable was larger than the one produced by the application of 150 ppm P, and the fungal effect on the nitrogen content was equivalent to that of this rate. The P and N content in the above ground plant parts increased in the plants not treated with Mycorrhiza as the rate of phosphate fertilizer increased. As expected, ordinary superphosphate produced a larger effect than phosphate rock (Figure 2a and c). In plants treated with Mycorrhiza, the application of P did not produce a significant effect on N content, but considerable increases did occur in the content of phosphorus. Within the group of plants fertilized with phosphate rock, the fungal activity and the phosphate fertilizer had additional effects over phosphorus content in the above ground biomass. In the experimental units treated with superphosphate, the 50 ppm application in the presence of fungus had a synergistic effect on this parameter. However, when the phosphorus level increased there was negative interaction, similar to that observed in the previous variables.
Figure 2. (A) Effect of the interaction of Mycorrhiza x P source x rate of P2 O5 fertilization over P content in above ground biomass of L. leucocepnala; (B,C, & D) Effect of interactions of Mycorrhiza x rate of P2 O5 application, Mycorrhiza x P source and Mycorrhiza x N source, respectively, over the N content in above ground biomass. +M = with Glomus intrarudices, M = without. OS = ordinary superphosphate, PR = phosphate rock. aerial part (mg/2 plants) p in aerial part (mg/2 plants) +M
As observed for the above-ground biomass, Rhizobium loti inoculation affected only the nitrogen content of the VAM-treated plants (Figure 2d). The 20 ppm N application had no effects in either of the aforementioned variables. Mvcorrhizal colonization. With the exception of the P application levels none of the factors studied had any effect on mycorrhizal colonization (Figure 3). As soon as the P content of soil was increased, total colonization gradually decreased. Both this variable and the intraradical vesicles frequency diminished by approximately 10 units when applying 50 ppm P to soil. This behavior exhibited little change with larger rates of P application. The percentage of arbuscules was not affected by factors under study. Discussion: Leucaena plants inoculated with Glomus intraradices exhibited greater capacity to uptake phosphorus from soil than non-inoculated plants. This difference produced large increases in the different growth parameters. The improvement in the phosphate nutrition of the plant favored the symbiosis with Rhizobium loti and consequently the fixation of atmospheric nitrogen. Nodulation was almost nil in the plants where phosphorus was not applied nor fungus inoculated. Dry weight of the nodules depend on the availability of this element for the plant. The nodules biomass was closely correlated to the production of dty matter (r = 0.85), nitrogen (r = 0.79) and phosphorus (r = 0.81) contents in the aerial part. This explains the positive results obtained with double inoculation (G. intraradices + R. loti) over the dry weight and nitrogen content in the aerial part. Figure 3. Effect of P7 O5 rate on Glomus intraradices root colonization of L. leucocephala. 0 50 100
The response to phosphate fertilization in plants without endomycorrhiza showed, in general, a linear trend. In some cases it appeared to adopt a pattern similar to a sigmoid curve, typical of nutrient-deficient soils (Volke, 1981). Nevertheless, VAM inoculation had the same effect as the highest rate of P fertilization (150 ppm P). This indicates that leucaena is a species of high mycorrhizal dependence in soils as those utilized in this research and its atmospheric nitrogen fixation potential can be efficiently utilized by means of double symbiosis (rhizobium + glomus). The endophyte s effect on the plant s nutrition and growth was reduced as the phosphorus content in the soil increased. This was due to the depressing effect of this element on VAM colonization. The linear correlation coefficient between this variable and the phosphorus content in the aerial part was negative (r = -0.531; inverse relations also were observed with the root (r = -0.56) and aerial part (r = - 0.3) dry weights. This indicates that the reduction in fungal colonization was related to a larger growth, and it occurred precisely in the plants that had the best phosphorus nutrition. As the availability of phosphorus increased, the plant reduced the endophyte presence in the radical system ana began substituting the biological with the mineral source, possibly with a reduced metabolic cost. The high response to VAM inoculation also is attributable to the initial low P content of the soil. The synergistic effect observed in the treatment with 50 ppm P (ordinary supeijvhosphate) shows that in P-deficient soils it is possible to reduce P fertilization by VAM inoculation and maintain a higher productivity level than could be obtained by applying either of factors independently. This would allow the most efficient utilization and significant savings on fertilizer. In P fertilization tests in acid soils or soils with high P fixing capacity, high rates of this element are frequently required to achieve yield increases. Such results are commonly explained in terms of the physical and chemical characteristics of the soil environment. Our data, however, indicate that the mycorrhizal symbiosis may have a significant influence on this type of response and the suppression of mycorrhizal fungi may generate a need for more fertilization. As a result of our research we may infer that whatever is the response to phosphorus application, under natural conditions the mycorrhizal symbiosis may have an important role in leucaena growth, thereby suggesting it should be taken into account in future studies of test for soil productivity. References: Gardezi, A.K., R. Ferrera-Cerrato, and V. Lara-Fernandez. 1988. Effect of the double inoculation of Rhizobium sp and V-A endomycorrhizae on Acacia cyanophylla in an andosol in Mexico. NFTRR 6:31-33. Lara-Fernandez, V. and R. Ferrera-Cerrato. 1986. Study of the vesicular-arbuscular endomycorrhizal Leucaena leucocephala symbiosis. LRR 7:94-96. Moreno, R.A. 1981. Eficiencia de cepas de rhizobium y efecto de P, Mo, Fe, Co y encalado en la nodulacion y produccion de biomasa de Leucaena leucocephala en suelos acidos de Huimanguillo, Tabasco. MS Thesis, Colegio de Postgraduados, Chapingo, Mexico. Munns, D.N. and Mosse, B. 1980. Mineral nutrition of legume crops, p 115-125. In RJ. Summerfield and A.H. Bunting (eds.) Advances in Legume Science. University of Reading, England. Volke, H.V. 1981. Estimacion de funciones de regresion en experimentos con fertilizantes y densidad de plantas con fines de determinacion de optimos economicos. Centro de Edafologia, Colegio de Postgraduados. Chapingo, Mexico. 65 p.