Pacific Agriculture and Natural Resources

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1 Pacific Agriculture and Natural Resources Evaluation of controlled-release fertilizer and abuscular mycorhizae inoculation for the production of Leucaena leucocephala container seedlings in a soil-based medium ADEL H.YOUKHANA 1 * AND MITIKU HABTE 1 Department of Natural Resources and Environmental Management, College of Tropical Agriculture and Human Resources, University of Hawaii at Manoa. Department of Tropical Plant and Soil Sciences, College of Tropical Agriculture and Human Resources. University of Hawaii at Manoa. Abstract: Two successive greenhouse experiments were undertaken to determine the effect of rate and method of application of the controlled release fertilizer (CRF) osmocote TM on arbuscular mycorrhizal activity in tree seedlings using Leucaena leucocephala var K3 as an indicator species. In the first experiment, arbuscular mycorrhizal fungal colonization of seedlings was significantly curtailed if osmocote was incorporated into soil at rates exceeding 1 g kg -1. This adverse effect was appreciably reduced by increasing the rate and intensity of irrigation and by placing osmocote on the surface of soil rather than incorporating it into the soil. In soil not amended with osmocote, AM fungal effectiveness measured as P content of Leucaena-K3 pinnules was not expressed. The highest level of AM fungal effectiveness was observed if osmocote was mixed with soil at the rate of 1 g kg -1 or if placed on the surface of soil at the rate of 1- g kg -1. Dry matter was significantly increased by mycorrhizal inoculation at all levels of osmocote except at the highest rate of incorporated osmocote. However, the effect observed at osmocote application rate of 1 g kg -1 was either superior or equal to those observed at the higher rates of osmocote application. Our data demonstrate that robust seedlings well colonized by AM fungi can be produced by mixing osmocote or placing it on the surface of soil at the rate of 1 g kg -1. Key words: Arbuscular mycorrhizal fungi, controlled release fertilizer, electrical conductivity, Glomus aggregatum, Osmocote TM, phosphorus, pinnule, soil solution, Leucaena leucocephala var K3. Introduction There is a growing interest to integrate arbuscular mycorrhizal (AM) technology in plant production regimes (Allen et al., 3; Jeffries et al., 3; Corkidi et al., 5; Meddad-Hamza et al., 1). The most cost-effective means of applying the AM fungal technology is to establish the fungi on plant roots at the nursery and to subsequently plant the seedlings in the field (Jeffries et al., 3; Pomper et al., 3). Arbuscular mycorrhizal fungi perform numerous functions that can influence pre-transplant and posttransplant seedling health and productivity (Jeffries et al., 3; Habte, ). However, there are no standardized protocols for producing tree seedlings well colonized by AM fungi under nursery conditions, since the procedures normally employed in these operations are not designed for mycorrhiza-dependent growth of plants (Peters and Habte, 1; Linderman and Davis, ). The growing mediums used for raising seedlings in nurseries are varied, and the nutrient levels and watering regimes employed are generally not conducive for mycorrhiza formation and function. In many places, peat is used as the matrix for raising seedlings while in many other places, soil is the medium of choice because of the absence of natural deposits of peat and the cost associated with importing it from elsewhere. The ability of AM fungi to reduce the external P requirement of associated seedlings has an important environmental benefit, as evidenced in a soil-based medium (Onguene and Habte, 1995) and in peat-based medium (Peters and Habte, 1). In both of these works, a rapidly available P source was employed, and the levels of other nutrients had to be adjusted on a regular basis during the growth of the seedlings. From the standpoint of environmental concerns and ease of management, control ease fertilizers may be preferred as nutrient sources (Carpio et al., 5). However, the rates of these nutrients to be applied for mycorrhiza-dependent growth of seedlings, particularly for seedlings raised on soil-based matrices are not clearly defined. The primary aim of the current work was to determine the method and rate of *Corresponding author, adel@hawaii.edu 1 Vol. 3: 1-7

2 Evaluation of controlled-release fertilizer and abuscular mycorhizae inoculation for the production of Leucaena leucocephala container seedlings in a soil-based medium application of the control ease fertilizer osmocote TM required for the production of tree seedlings well colonized by AM fungi using L. leucocephlala var. K3 as an indicator tree species. Materials and Methods The soil used in this investigation was a subsurface sample (1-5 cm) of an Ultisol (Clayey, Oxidic, Isothermic, Typic Kandihumult, Leilehua series) with a ph of. (1: soils-water suspension) and an initial soil solution P concentration of.9 mg l -1. Seeds of Leucaena leucocephala (Lam.) De Wit var. K3 were scarified in concentrated H SO for 3 min and rinsed with sterile water four times before they were germinated in moistened and sterilized paper towel at 3 o C for 3 days. Uniformly germinated seeds were selected for planting in D1 plastic Deepots (Stuewe and Sons, Inc, Corvallis, Oregon), containing g (dry wt) of soil per Deepot. Osmocote TM (19--1, macronutrient) was applied on the surface of the soil or incorporated into the soil at five rates of,1,, 3 and 8 g kg -1 of soil. Treatments consisted of two levels of mycorrhizal inoculation (not and ), five levels of fertilization, and two methods of osmocote application. Treatments were arranged on racks in a completely randomized design with three replicates per treatment. Mycorrhizal inoculation of some of the soil in the Deepots was achieved by thoroughly mixing the contents of each Deepot with 1g of a crude inoculum of Glomus aggregatum consisting of sand, spores, bits of hyphae and pieces of infected roots. The crude inoculum contained infective propagules g -1. The un soil received 1g of the inoculum carrier consisting of calcined montmorillonite (Turface TM ) and crushed basalt (Mansand TM ) in a ratio of :1 by volume. Micronutrients were added as 8 ml of Hoagland s micronutrient solution (Hoagland and Arnon, 195). The germinated leucaena seeds were planted in 7 cm deep depressions made at the center of the Deepot. Moisture was supplied to the plants by means of overhead sprinklers. The experiment was done twice. In the first experiment, the sprinklers were run twice daily 5 minutes irrigation -1. A second study was conducted to if the detrimental effect on seedlings of the higher osmocote levels initially observed were related to salt accumulation and if it could be alleviated by increasing the irrigation duration (three times daily for 1 minutes). Plants were grown in the greenhouse under natural light from September 1 to November 1, 5 (first experiment) and from December, 5 to March, (second experiment). The development of AM fungal effectiveness was monitored by measuring P content of pinnules from the youngest fully expanded leaves of leucaena plants (Habte, 199). Starting at two weeks after emergence, pinnule P content was determined at weekly intervals. Pinnules were dried for 3 hours at 7 o C, after which time they were placed in glass test tubes and dry-ashed by placing the test tubes in a muffle furnace at 5 o C for 3 hours. Phosphorus content of the ashed samples was determined by the phosphomolybdate blue method (Murphy and Riley, 19). Leachate samples were collected 1, 1, 8, 35, and days after emergence. They were collected by enclosing the bottom openings of the Deepots with plastic bags. Electric conductivity (EC) of the leachate samples was determined by means of a Cole-Parmer Solution Analyzer (Cole-Parmer Instrument Co., Chicago, IL) fitted with a conductivity dip cell. Plants were harvested at and 7 days after planting for the first and the second experiments, respectively. Shoots were cut at the soil line and dried at 7 o C for (7) hrs for dry matter determination. Arbuscular mycorrhizal fungal colonization of roots was determined by the grid-line intersect method (Giovannetti and Mosses, 198) after staining roots as outlined by Habte and Osorio, (1). Analysis of data was carried out using a SAS procedure (SAS Institute Inc., 199) and treatment comparisons were made using Least Significant Difference (LSD) if F values were significant. Tab.1: Effect of osmocote rate, method of application, and arbuscular mycorrhizal inoculation on dry matter yield of L. leucocephala and colonization of its roots by G. Aggregatum in first experiment *. Osmocote (g kg -1 ) Dry matter yield (g) AM fungal colonization % Surface applied.j.7ij a 1.3ij 1.8a 1bc.1hij 1.5abc 58ab 3 1.ab 1.9bcd 5abc 8.8hij 1.ab 37c Incorporated in soil.f.7f a 1.7gh 1.55abc 39c.83fg 1.ed 19d 3.3hij 1.3cde 9d 8.5ghi.79fg 8d *Means followed by the same letter are not significantly different at the 5% level. Results and Discussion Results First experiment: AM fungal colonization of roots Roots of plants grown in soil not with G. aggregatum showed no evidence of AM fungal Vol. 3:1-7

3 Adel H. Youkhana and Mitiku Habte 3 colonization (Table 1). Arbuscular mycorrhizal colonization of roots of plants grown in soil with G. Aggregatum was highest in soil not amended with osmocote. Colonization on plant roots grown in pots where osmocote was surface-applied ranged from 37-58%. Thirty nine percent of the root length of plants grown in soil into which osmocote was incorporated at the rate of 1 g kg -1 was colonized by AM fungus. Arbuscular mycorrhizal colonization was severely reduced by the higher rates of osmocote tested. Pinnule (P) content: In soil not amended with osmocote, AM fungal inoculation did not influence AM symbiotic effectiveness monitored as P content of leucaena pinnules (Fig. 1). If osmocote was applied on the soil surface, AM symbiotic effectiveness was expressed at all rates of osmocote application, especially in plant grown in soil amended with 1 g kg -1 osmocote. Mycorrhizal inoculation effect declined progressively with increasing osmocote rate. Similarly, for treatments where osmocote was incorporated, AM symbiotic effectiveness was also most expressed for the 1 g kg -1 rate of osmocote declined progressively thereafter until no symbiotic effectiveness was detected at the highest osmocote rate (Fig. ). P pinnule gkg -1 osmocote 1 gkg -1 osmocote 3 gkg -1 osmocote Col 3 vs Col Col 3 vs Col gkg -1 osmocote 5 gkg -1 osmocote Fig.1: Influence of surface application of osmocote on P content of pinnules of mycorrhizal and mycorrhiza-free L. leucocephala in first experiment. Dry matter yield In soil not with AM fungus, surface application of osmocote stimulated growth of L. leucocephala seedlings significantly only at the fertilizer rate of 3 g kg -1 (Table 1). In soil with G. aggregatum, surface applied osmocote stimulated dry matter yield significantly at all rates of fertilization. Incorporation of osmocote into soil stimulated dry matter yield of L. leucocephala at all rates of application except at 3 g kg -1 in the soil not with G. aggregatum. Arbuscular mycorrhizal inoculation enhanced seedling growth at all rates of osmocote; but seedling growth at highest osmocote concentration was inferior to that observed at the lower levels. For both surface applied and incorporated osmocote, AM inoculation effect observed at osmocote application rate of 1 g kg -1 was either comparable or superior to that observed at the higher application rates. P pinnule-1 gkg -1 osmocote 1 gkg -1 osmocote 3 gkg -1 osmocote Col vs Col Non gkg -1 osmocote 8 gkg -1 osmocote Fig. : Influence of incorporating osmocote into soil on the P content of pinnules of mycorrhizal and mycorrhiza-free L. leucocephala in first experiment. Second experiment: AM fungal colonization of roots: Roots of plants grown in soil not with G. aggregatum showed no evidence of AM fungal colonization (Table ). Arbuscular mycorrhizal colonization levels of roots of plants grown in soil ranged from -83% if osmocote was surface-applied. Roots of plants grown in soil amended with 1-3 g of osmocote kg -1 had AM fungal colonization levels in excess of 7%. The level of AM fungal colonization for roots of plants grown in soil Vol. 3: 1-7, 11

4 Osmocote rate (g kg -1 ) Evaluation of controlled-release fertilizer and abuscular mycorhizae inoculation for the production of Leucaena leucocephala container seedlings in a soil-based medium amended with the highest level of osmocote was significantly lower than all other rates. Table : Effect of osmocote rate and method of application on arbuscular mycorrhizal fungal colonization and growth of L. Leucocephala in the second experiment*. pinnules revealed that AM fungal inoculation had no Dry matter yield (g) AM fungal colonization % significant influence in soil that was not fertilized (Fig. 3). Osmocote amendment stimulated P content de.ab 5cd 5.33cd.3ab 5c * Means followed by the same letter within a measured variable are not significantly different at the 5% level. gkg -1 osmocote Pinnule (P) content: Arbuscular mycorrhizal symbiotic effectiveness measured in terms of P content of L. leucocephala significantly at all rates irrespective of method of application, although to a lesser degree at rates higher Applied on the soil surface than g kg -1 (Fig. 3 and ). The highest P content was associated with osmocote amendment of 1 and g.ef.3f d kg -1.for both methods of application and these values 1.38ef 5.a 83a were noted at 35 to 58 days from planting, respectively..5ef 5.33a 7ab The patterns of symbiotic effectiveness observed if ef.1ab 71b osmocote was placed on the soil surface was similar to that observed if the fertilizer was incorporated into soil ef.5ab 58c However, the relative effect of Incorporated into soil mycorrhizal inoculation was less pronounced if the.3f.7f d fertilizer was incorporated into soil than if it was placed 1 1.8ef.53ab 8ab on the surface of the soil (Fig 3 and ). 1.75de.5ab 7ab gkg -1 osmocote 1 gkg -1 osmocote gkg -1 osmocote P pinnule -1 1 gkg -1 osmocote 3 gkg -1 osmocote gkg -1 osmocote 8 gkg -1 osmocote Fig. 3: Influence of surface applied osmocote on P content of pinnules of mycorrhizal and mycorrhiza-free L. leucocephala in second experiment. P pinnule -1 3 gkg -1 osmocote gkg -1 osmocote Fig. : Influence of incorporating osmocote into soil on the P content of pinnules of mycorrhizal and mycorrhizafree L. leucocephala in second experiment. Dry matter yield: The lowest rate osmocote regardless of application methods increased non-mycorrhizal seedling growth Vol. 3:1-7

5 Adel H. Youkhana and Mitiku Habte 5 significantly. Higher rates either did not improve seedling size or yielded smaller seedlings (Table ). Mycorrhizal inoculation stimulated seedling growth significantly at all rates of osmocote. However, differences among osmocote rates were not significantly different. The best mycorrhiza-dependent seedling growth was associated with osmocote rates of 1-3 g kg-1, the highest rate of osmocote tested yielding somewhat inferior mycorrhizal seedlings. Electrical conductivity (EC): Electric conductivity (EC) monitored as a function of time during the second study revealed that at least during the initial stages of seedling growth plants were exposed to relatively high salinity levels at the higher osmocote, especially if osmocote was incorporated into the soil (Table 3). Tab.3 Electric conductivity (EC) of leachates of growth medium at different times from emergence. Osmocote rate (g kg -1 ) Days after emergence (mmhos cm -1 ) I NI I NI I NI I NI I NI Surface applied Incorporated LSD I = with G. aggregatum; NI = ; Surface applied = Osmocote applied on the surface of the growth medium; Incorporated = Osmocote thoroughly mixed with the growth medium. Discussion The extent to which roots were colonized by AM fungi, symbiotic effectiveness measured in terms of foliar P content and shoot dry matter yield were significantly influenced by AM inoculation and rate and method of osmocote application. Arbuscular mycorrhizal colonization of roots observed in the absence of osmocote in the first experiment was higher than that observed in the second experiment. This is probably due to the higher irrigation intensity and frequency in the latter experiment which leached P in the soil solution, thereby reducing it to a concentration which was less than optimal for AM formation (Habte and Manjunath, 1987). In the absence of osmocote, there was no correlation between AM fungal colonization and AM fungal symbiotic effectiveness measured as pinnule (P) content or dry matter yield in both experiments. Arbuscular mycorrhizal colonization levels as well as AM symbiotic effectiveness are regulated by tissue (P) concentration which in turn is closely related to soil solution (P) concentration (Habte and Manjunath, 1987). However, AM formation is less sensitive to suboptimal solution (P) concentrations, and AM fungi often colonize host roots under extremely low (P) concentrations whereby they provide little or no benefit to the associated host or develop at the expense of the host (Habte, ). The lower AM fungal colonization level observed if the lowest rate of osmocote was incorporated into soil compared to that observed if the soil was not fertilized (first experiment) suggests that the (P) concentration in the soil solution was higher than the optimal concentration needed for AM fungal colonization. This inference is strengthened by significant reduction in AM fungal colonization observed with increasing rate of osmocote. However, the AM fungal colonization observed at the lowest osmocote rate was sufficient to yield seedling growth that was better or equal to that observed at higher osmocote rates and in the absence of osmocote. Applying osmocote on the surface of soil at the rate of 1 g kg -1 did not yield AM fungal colonization level that was significantly different than that observed if the fertilizer was incorporated into soil, although subsequent increments of surface applied osmocote did not result in the drastic reduction in AM fungal colonization noted if osmocote was incorporated into soil at rates in excess of 1 g kg -1. These differences are explained by the slow rate at which P from surface applied osmocote defused from the surface of the soil to the root zone. Seedling growth was not significantly improved by increasing the concentration of surface-applied osmocote above 1 g kg -1. We do not, therefore, recommend the use of osmocote rates higher than 1 g kg -1 for the production of mycorrhizal seedlings under conditions similar to those prevailing in our studies. Since growth of mycorrhiza-free L. leucocephala as well as pinnule P status in the absence of osmocote were consistently inferior compared to those observed in the presence of osmocote, it is tempting to conclude that osmocote had no inhibitory effect on L. leucocephala. This is a reasonable inference for surface applied osmocote. However, the relatively poor responses of mycorrhiza-free L. leucocephala to the higher rates of incorporated osmocote, particularly to the two highest Vol. 3: 1-7, 11

6 Evaluation of controlled-release fertilizer and abuscular mycorhizae inoculation for the production of Leucaena leucocephala container seedlings in a soil-based medium rates suggest that the material was toxic to the plant at these rates. It is evident from pinnule P data that P was readily available to the plant if osmocote was incorporated into soil than if it was applied on the surface of soil. We believe that the inhibitory effect of osmocote incorporated into soil may in part be due to the effect of salt build up on the host. AM fungi are known to be more tolerant to salinity than associated plants (Al-Karaki, ; Yeno-Melo et al., 3). This is supported by the fact that in the second experiment in which the duration of irrigation was deliberately increased to enhance salt leaching; mycorrhizal colonization and mycorrhizal effectiveness were enhanced or maintained over a wider range of osmocote rates irrespective of the method of osmocote application (Tables 1 and ; Fig 3 and ). These observations were further supported by the EC data which shows that even with the relatively longer duration of irrigation, there was some accumulation of salt during the initial phase of the development of seedlings and that salt build up was much more pronounced at higher osmocote concentrations, particularly if osmocote was incorporated into soil. Therefore, it is reasonable to assume that in the first experiment whereby the duration of irrigation was shorter than that in the second experiment, the adverse effect of salt accumulation would have been much greater than that detected during the second experiment. The concentrations of P added through surface applied osmocote were not sufficient for mycorrhiza-free growth of L. leucocephala since seedlings responded to AM fungal inoculation at all rates of surface applied osmocote and at all but the highest rate of incorporated osmocote in both experiments. That seedling growth was stimulated at very low AM fungal colonization levels in soil into which osmocote was incorporated at the rate of -3 g kg -1 is interesting. Although instances of such stimulations have been noted by other investigators (Carpio et al., 5), they are quite rare. The better overall growth of seedlings in the second experiment reflects the better light and moisture conditions and the lower salt load which prevailed during the duration of the study, compared to those that prevailed during the first experiment. In both experiments, mycorrhizal inoculation effect and seedling size were not improved by concentrations of osmocote in excess of 1 g kg -1 despite the widely differing mycorrhizal status of the test plants. Because plants rely on the mycorrhizal condition for nutrient uptake and growth, depending on their inherent ability to take up P from the soil solution, no one rate of osmocote is expected to serve as optimal for the production of robust mycorrhizal tree seedlings of all species (Habte and Manjuanth, 1991). Plant species that are marginally or moderately dependent on the mycorrhizal condition for nutrient uptake and growth tend to take up P readily at relatively low solution P concentrations and tend to accumulate high levels in their tissue which in turn can inhibit AM fungal colonization of their roots (Manjunath and Habte, 199). The optimum rate of osmocote for mycorrhizadependent growth of these species is likely to be lower than 1 g of incorporated osmocote kg-1 and we anticipate that the risk of inhibiting AM fungal colonization by excess P from osmocote in these species can be minimized by applying the fertilizer on the surface of the growth medium. The slow rate at which P defuses from the surface of the soil to the root zone will ensure that roots not be exposed to high concentrations of P which will inhibit AM fungal development. However, for those tree species that are very highly dependent on the mycorrhizal condition for nutrient uptake and growth such as L. leucocephala, 1 g of osmocote kg -1 appears to be near-optimal for sufficient mycorrhization and robust seedling growth. In situations in which it is necessary to keep seedlings in the nursery for durations much longer than the durations of our study, we recommend that supplemental osmocote be applied on the soil surface at the rate of 1 g kg -1 as needed. It is clear from our data that such a practice is not likely to cause inhibition of mycorrhizal colonization. Conclusion In the absence of osmocote, there was no correlation between AM fungal colonization and AM fungal symbiotic effectiveness measured as pinnule P content or dry matter yield in both experiments. The lower AM fungal colonization level observed if the lowest rate of osmocote was incorporated into soil compared to that observed if the soil was not fertilized. The highest level of AM fungal effectiveness was observed if osmocote was mixed with soil at the rate of 1 g kg -1 or if placed on the surface of soil at the rate of 1- g kg -1. Our data demonstrate that robust seedlings well colonized by AM fungi can be produced by mixing osmocote or placing it on the surface of soil at the rate of 1 g kg -1. Literature Cited Allen, E.B., Allen, M. F., Egirton, L., Corkidi, L. and Gomez- Pompa, A. 3. Impacts of early and late serial mycorrhizae during restoration in seasonal tropical forest. Ecological Applications 13: Al-Karaki, G. N.. Growth of mycorrhizal tomato and mineral acquisition under salt stress. Mycorrhiza 1: Caprio, L.A., Davies, F.T., and Arnold, M.A. 5. 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