Optimizing Solution P Concentration in a Peat-Based Medium for ...

Arid Land Research and Management, 15:359± 370, 2001 Copyright # 2001 Taylor & Francis 1532-4982 /01 $12.00 ‡.00

Optimizing Solution P Concentration in a Peat-Based Medium for Producing Mycorrhizal Seedlings in Containers

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S. M. PETERS M. HABTE Department of Tropical Plant and Soil Science College of Tropical Agriculture and Human Resources University of Hawaii Honolulu, Hawaii, USA An investigation was undertaken to test the hypothesis that amending peat to increase its P bu€ er capacity and optimizing the P concentration of the amended medium for mycorrhizal activity will enhance its usefulness for raising mycorrhizal seedlings of tree species. The approaches entailed mixing a small quantity of soil of high P adsorbing capacity with peat and constructing a P sorption isotherm for the medium in order to establish solution P concentration near-optimal to optimal for mycorrhization of seedlings. A P sorption isotherm based on incubating the medium with graded amounts of P at 50% of available water-holding capacity was developed. T arget solution P concentrations established using the approach enabled us to identify the optimal solution P concentration for mycorrhizal development on roots of our indicator plant L eucaena leucocephala grown in the medium. Arbuscular mycorrhizal colonization, host growth, and P status of L . leucocephala pinnules measured at target solution P concentrations ranging from 0.12 to 1.0 mg L ± 1 revealed that AM fungal activity and symbiotic e€ ectiveness was maximum at solution P concentration of 0.2 mg P L ± 1 . Medium solution P concentrations in excess of 0.2 mg L ± 1 tended to depress AM fungal colonization, but colonization level did not decline below 43% . Keywords AMF colonization, nursery, P bu€ er capacity, peat, pinnules, P sorption isotherm, seedlings, solution P, L eucaena leucocephala

Arbuscular mycorrhizal (AM) fungi form bene® cial association with roots of most cultivated and naturally occurring species of plants. Their positive e€ ects on plants are largely explained by the ability of the fungi to enhance the uptake of P and other di€ usion-limited nutrients. The fungi are also known to protect associated plants against pathogens, and adverse environmental conditions such as drought and salinity, and to promote soil aggregation (Bethlenfalvay 1992; Schreiner and Bethlenfalvay 1995). Because the fungi cannot be multiplied in laboratory media, inocula are generally produced in sand, soil, or sand-soil matrix in the presence of a suitable host plant. Inocula produced in this manner are crude and bulky, and do not lend themselves to direct application to extensive areas of land. The best prospect for Received 18 January 2001; accepted 5 March 2001. Journal series No. 4573, College of Tropical Agriculture and Human Resources, University of Hawaii. Address correspondence to Dr. M. Habte, Department of Tropical Plant and Soil Science, University of Hawaii, 3190 Maile Way, Honolulu, Hawaii 96822, USA. e-mail: mitiku@ hawaii.edu

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application of the fungi, therefore, lies with species of plants that are normally transplanted, in which case thousands of mycorrhizal seedlings could be raised in small areas in the nursery for subsequent planting to large areas of land. The containerized nursery production practices that are currently prevalent in the nursery industry are peat-based . While peat has several desirable properties for growing seedlings, namely its light weight, high water-holding capacity, and large air-® lled pore spaces (Brady and Weil 1999) it is not a good medium for the development of arbuscular mycorrhizas, even without the current practice of heavy fertilization (Biermann and Linderman 1983; Pedersen et al. 1991). Our own experience with commercially available peat-based media has been that they are either very unpredictable or unsuited for raising mycorrhizal seedlings (M. Habte, unpublished data). A number of attempts have been made to evaluate the e€ ectiveness of various additives in order to make peat conducive to mycorrhizal development (Biermann and Linderman 1983; Johnson and Hummel 1986; Pedersen et al. 1991). However, the multiplicity of materials, approaches, and fertilization practices involved have not led to the development of a standardized approach for raising vigorously growing mycorrhizal seedlings. Soil-based media are well suited for mycorrhization of seedlings (Onguene and Habte 1995), but their weight and relatively low waterholding capacity makes them unsuited for raising large numbers of seedlings. We hypothesize that the inferiority of peat as mycorrhization medium is related to its low P bu€ er capacity, and this could be overcome by adding to it a small quantity of soil with high P bu€ er capacity (high P adsorbing capacity) and then optimizing P content of the mixture for AM development. The objective of the current study, therefore, was to determine the solution P concentration that is optimal for mycorrhization of seedlings in a peat-based medium after elevating its P bu€ er capacity.

Materials and Methods Medium for Raising Seedlings The medium used was a mixture of mineral soil and organic material combined in a ratio of one part mineral soil to three parts organic material on a dry weight (dw) basis. The mineral soil used was a subsurface sample (10± 25 cm depth) from the Leilehua series (clayey, oxidic, isothermic typic kandihumult) (C. Smith, personal communication). The organic material used was Canadian sphagnum peat moss (Premier Horticulture Inc., Red Hill, Pennsylvania) (abbreviated as PSPM). The soil was ground to pass through a 1± mm sieve and the PSPM was crushed to pass through a 4± mm sieve. The sieved materials were thoroughly mixed in the ratio indicated above by rolling them on brown paper side to side and front to back several times. Dolomite (C. Brewer Environmental Company, Honolulu, Hawaii) was added according to a liming curve to bring the PSPM-soil mixture to a pH of 6.5. After the Dolomite was incorporated into the medium, the moisture content of the mixture was brought up to maximum water-holding capacity and then allowed to equilibrate for at least two weeks. The growth medium was then placed in a plastic tub and sterilized in an autoclave at 205 kPa and 1218C for 60 min two times at an interval of one day. Preparation of Growth Medium A phosphorus sorption isotherm developed as described by Fox and Kamprath (1970) was compared to one based on incubating the medium with graded amounts of P at approximately 50% water-holding capacity. The latter approach will be designated as the incubation method or approach hereafter. Due to the extraordinarily high solution P concentrations measured when we used the Fox and

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Kamprath (1970) approach, the P sorption isotherm developed by the incubation method was chosen for establishing target solution P concentrations in the test medium. The incubation method di€ ers from the Fox and Kamprath (1970) method primarily in the moisture status of the medium during incubation and in the length of incubation period required to attain equilibration. The incubation method consisted of dissolving various amounts of KH2 PO4 in enough de-ionized water to wet 200 g portions of the growth medium to 50% of its water-holding capacity (AWC). The medium was then thoroughly mixed in a Zip-loc bag (Presto Products Company, Appleton, Wisconsin) and then transferred into a screw-capped jar which contained a 1.5 cm layer of water agar (1.5% agar, w/v) in order to maintain the medium at the adjusted moisture content of approximately 50% AWC. The solution P concentration of the medium was measured periodically by shaking a 0.5 g portion of the medium in 30 mL of 0.01 M CaCl2 in a centrifuge tube for one hour on a reciprocating shaker. After the suspension was centrifuged for 10 min at 10,000 rpm in a Sorvall super speed centrifuge (Dupont Instruments, Claremont, California), the supernatant was ® ltered through a Fisher P8 ® lter paper and the P concentration of the ® ltrate was determined spectrophotometricall y (Murphy and Riley 1962). A phosphorus sorption isotherm was constructed using P concentration values observed at day 29. Based on the P sorption isotherm, target P concentrations were established by dissolving appropriate amounts of KH2 PO4 in P free Hoagland’s solution (Hoagland and Arnon 1950). The P-free solution was based on the amount of nutrients that is su cient to support seedlings for 42 days. This was calculated to be 80 mL of Hoagland’ s solution containing six times the normal concentration of nutrients for each 250 g portion of medium. Su cient water was added to the solutions in order to raise the moisture content of the PSPM-soil medium to 50% of maximum water-holding capacity. The solutions were added into the medium one day before planting and the medium was throughly mixed in a Ziploc plastic bag by massaging. The target solution P concentrations were 0.12, 0.2, 0.4, 0.6, 0.8, and 1.0 mg L ± 1 . Inoculation with (AMF) was achieved by adding crude inoculum of Glomus aggregatum Schenck and Smith emend Koske, consisting of sand, extramatrical spores and sporocarps, bits of hyphae, and infected root segments at the rate of 200 g per kilogram of medium. The inoculum was freshly produced as previously described (Habte, Zhang and Schmitt 1999) and contained average 2.4 viable propagules g± 1 . The uninoculated medium received 200 g of sterilized sand kg ± 1 of medium and a ® ltrate of the crude inoculum obtained by suspending 10 g of the crude inoculum in 100 mL of deionized water and passing it through a Whatman No.1 ® lter paper. Ten mL of the ® ltrate was added to each 250 g portion of PSPMsoil mixture along with the Hoagland’s solution. The crude inoculum and the sterilized sand were mixed with their respective growth medium prior to nutrient amendment and planting. Mixing was achieved by spreading the medium and inoculum or sand on a brown paper and rolling the paper front to back and side to side. Seed Preparation and Planting L eucaena leucocephala (Lam.) De Wit var K636 (AgroForester Tropical Seeds, Holualoa, Hawaii) was the indicator plant used. Seeds of the legume were scari® ed by immersion in concentrated sulfuric acid for 30 min and then rinsing them four times with sterilized water. Seeds were germinated in sterilized glass Petri dishes lined with ® lter papers. The Petri dishes and ® lter papers were sterilized by microwaving for ® ve minutes after moistening the ® lter paper. Because the ® lter papers became dry during microwaving, they were rewetted with sterile deionized water. Seeds were then placed in them, and the plates were incubated in the dark at 288C for two days. Uniform seeds were selected for planting in D16 plastic Deepots (Stuewe and Sons, Inc, Corvallis, Oregon) of 28 cm £ 28 cm £ 18 cm. One seed was planted per

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Deepot. Uniformity was based on seed size and extent of radicle growth. A one-inch deep depression was made with forceps at the center of the medium contained in the Deepots and the seeds were then planted in the depressions with the radicle pointing downward. The seeds were then lightly covered with the growth medium. After emergence, treatments were arranged in a randomized complete block design on glasshouse benches with ® ve replicates per treatment. Plants were grown under natural light (21851’ N and 156822’ W). They were watered as needed to maintain the medium at approximately maximum water-holding capacity.

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Weekly Sampling and Harvest Starting two weeks after emergence, plant height and pinnule P content were determined at regular intervals. The P content of pinnules was determined as described by Habte (1992). The third pinnule of the youngest fully expanded leaf was removed for this purpose. Plants were harvested 42 days after planting. Measurements taken at harvest were shoot dry weight, plant height, P content of pinnules, the proportion of root colonized by the arbuscular mycorrhizal fungus, and solution P concentration of the medium. Shoots were dried for 48 hours at 708C before they were weighed. Plant height was measured from the base of the plant to the top of the growing tip, and P content of pinnules was determined as described above. Roots were cleared with 10% KOH and stained using a modi® cation of the procedures described by Kormanik, Bryan, and Shultz (1980). The modi® cation (Habte and Soedarjo 1995) involved using 0.15% acid fuchsin in a lactic acid-glycerol solution instead of a 0.015% acid fuchsin solution and standing at room temperature for 48 h instead of autoclaving for 15 min. The proportion of root colonized by AMF was determined by the grid-line intersect method (Giovannetti and Mosse 1980). Solution P concentration of the growth medium was determined spectrophotometricall y as described above in the monitoring of solution P concentration by the incubation method. Statistical Analysis Data were analyzed by ANOVA with arcsine transformations made before AMF colonization data were analyzed. The statistical software used was Statistix for Windows (Analytical Software, Tallahassee, Florida, 1996). When the F test was signi® cant, the least signi® cant di€ erence (LSD) was used to compare means at the P µ 0:05 level.

Results The results of the incubation approach for P sorption determination showed that medium solution P concentrations were high three days after P application but steadily declined thereafter, leveling out after 29 days of incubation (Figure 1). The P sorption isotherm was constructed using solution P concentrations values observed at this time (Figure 2). This P sorption isotherm was then used to determine the amount of P to be added to the growth medium in order to establish desired target solution P concentrations. The P sorption isotherm developed by the Fox and Kamprath (1970) method resulted in extraordinarily high solution P concentrations that are not likely to be observed under normal medium moisture regimes. In the growth experiment, solution P concentrations of the inoculated and uninoculated medium measured at planting were not di€ erent from each other (Table 1). After 42 days of L . leucocephala growth, the solution P concentrations of the medium supporting mycorrhizal L . leucocephala were lower than that supporting nonmycorrhizal plants (Table 1). This di€ erence was most pronounced starting at

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FIGURE 1 In¯ uence P amendment of the peat-based medium on the concentration of P attained in solution as a function of time.

FIGURE 2 Phosphorus adsorption curve at 29 days of the peat-based medium developed using our incubation approach. target solution P concentration of 0.6 mg L ± 1 whereby the medium supporting nonmycorrhizal plants had nearly twice as much P in solution as the medium supporting mycorrhizal L . leucocephala. None of the plants grown in the uninoculated medium had evidence of AMF colonization (Table 2). Mycorrhizal colonization of roots of L . leucocephala grown in the inoculated medium was maximum at target solution P concentration of 0.2 mg L ± 1 (Table 2, Figure 3). AMF colonization percentage declined as solution P concentration increased above this concentration but the percentage of colonization was not lower than 43%, a percentage which was observed at the highest medium

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TABLE 1 Medium solution phosphorus concentrations at the time of planting and after 42 days of growth of nonmycorrhizal and mycorrhizal L eucaena leucocephala Medium phosphorus (mg L ± 1 )

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Phosphorus added (mg kg ± 1 ) 0 80 120 150 180 210

Target medium solution P (mg L ± 1 ) 0.13 0.2 0.4 0.6 0.8 1.0

After growth of L . leucocephala UI I

At planting UI I 0.12 j 0.97 i 1.93 g 2.66 e 3.68 c 4.54 a

0.17 j 0.96 i 1.78 h 2.49 f 3.29 d 4.33 b

0.11 i 0.18 h 0.35 f 0.52 c 0.67 b 0.93 a

0.10 i 0.13 i 0.20 h 0.26 g 0.40 e 0.47 d

UI, uninoculated; I, inoculated. Means with the same letter in a column or row at a particular time of observation are not signi® cantly di€ erent at the P µ 0:05.

TABLE 2 E€ ect of mycorrhizal inoculation and medium solution phosphorus concentration on dry matter yield and arbuscular mycorrhizal fungal colonization of L eucaena leucocephala Phosphorus added (mg kg ± 1 ) 0 80 120 150 180 210

Target medium solution P (mg L ± 1 ) 0.13 0.2 0.4 0.6 0.8 1.0

Dry matter yield Shoot (g) UI I 0.21 0.45 0.54 0.60 0.66 0.79

f e de cd bc a

0.18 0.69 0.63 0.66 0.65 0.73

f abc bcd bcd bcd ab

AMF colonization (% root length) UI I 0 0 0 0 0 0

0 62.9 61.9 51.3 46.4 42.7

d a ab bc c c

UI, uninoculated; I, inoculated. Means with the same letter in a column or row of a particular time of sampling are not signi® cantly di€ erent at the P µ 0:05.

solution P concentration (Table 2). Inoculated plants growing in the medium that did not receive P were not colonized by AMF (Table 2). The pinnule P concentrations of nonmycorrhizal plants declined linearly with time at all target solution P concentrations tested (Figure 4). In contrast, pinnule P concentrations of mycorrhizal plants peaked at 35 days after planting (DAP), except for those of plants grown at 1.0 mg P L ± 1 which peaked at 28 DAP (Figure 4). After reaching peak values, pinnule P concentrations of mycorrhizal L . leucocephala dropped sharply at all target solution P concentrations of the medium except at the 0.8 and 1.0 mg P L± 1 , which remained relatively constant throughout the growth period (Figure 4). The time at which di€ erences in the pinnule P content of mycorrhizal and nonmycorrhizal plants became signi® cant varied depending on medium solution P concentration (Figure 4). This e€ ect was observed as early as 22 DAP at target solution P concentrations of 0.6 mg L ± 1 , and as late as 35 DAP at target P concentration of 0.8 mg L± 1 . The e€ ect of inoculation remained signi® cant until the termination of the experiment except for plants grown at solution P concentration of 0.6 mg L ± 1 , in

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FIGURE 3 In¯ uence of medium solution P concentration on arbuscular mycorrhizal fungal colonization of L eucaena leucocephala roots. Regression is signi® cant at ¬ = 0:01.

FIGURE 4 In¯ uence of medium solution P status on the concentration of P in pinnules of mycorrhizal and nonmycorrhizal L eucaena leucocephala. Bars represent LSD ( ¬ = 0:05).

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S. M. Peters and M. Habte TABLE 3 E€ ect of mycorrhizal inoculation and medium solution phosphorus concentration on pinnule P content of L eucaena leucocephala 42 days after planting. Pinnule P Content

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Phosphorus added (mg kg ± 1 ) 0 80 120 150 180 210

Target medium solution P (mg L± 1 ) 0.13 0.2 0.4 0.6 0.8 1.0

Concentration (g kg ± 1 ) UI I 0.86 f 0.68 f 0.84 f 1.33 de 1.03 ef 1.43 cde

0.61 1.29 1.80 1.67 2.12 2.36

f de bc cd ab a

UI, uninoculated; I, inoculated. Means with the same letter in a column or row of a particular measurement are not signi® cantly di€ erent at the P µ 0:05.

which case di€ erences in pinnule P concentration of mycorrhizal and nonmycorrhizal plants ceased to be signi® cant at day 42 (Figure 4, Table 3). Pinnule P concentration was lowest for both mycorrhizal and nonmycorrhizal plants if they were grown in the medium not amended with P. These values were 0.12 g kg ± 1 and 0.7 g kg± 1 for the mycorrhizal and nonmycorrhizal plants, respectively. The highest pinnule P concentrations observed were 2.8 g kg ± 1 for mycorrhizal plants and 2.4 g kg ± 1 for nonmycorrhizal ones. Mycorrhizal inoculation e€ ect (MIE) measured as the ratio of pinnule P concentration of inoculated and uninoculated L . leucocephala was maximum at day 28 for plants grown at medium P concentration of 1.0 mg L ± 1 and at day 35 for plants grown at the other solution P concentrations (Figure 4). Mycorrhiza inoculation e€ ect was well correlated with root colonization (Figure 5).

FIGURE 5 Relationship between AMF colonization and mycorrhiza inoculation e€ ect (MIE) measured as the ratio of the pinnule P content of inocualated (I) and uninoculated (UI) L eucaena leucocephala grown in the peat-based medium. Based on measurements taken at the time of peak mycorrhizal activity. Regression is signi® cant at ¬ = 0:01.

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FIGURE 6 E€ ect of medium solution P concentration on shoot dry weight of L eucaena leucocephala. Bars represent LSD ( ¬ = 0:05). Shoot dry matter yield data collected at the time of harvest revealed that the e€ ect of mycorrhizal inoculation was most pronounced at target solution P concentration of 0.2 mg L ± 1 (Figure 6). This e€ ect diminished as medium solution P concentration increased, growth of mycorrhizal plants appearing to be depressed at solution P concentration of 1.0 mg L± 1 (Figure 6). Above target solution P concentration of 0.2 mg L ± 1 , the shoot dry matter yields of mycorrhizal plants were not statistically di€ erent from each other (Table 2). However, shoot dry matter yields of nonmycorrhizal plants increased linearly with increases in solution P concentration, reaching dry weight values comparable to mycorrhizal plants at target P concentration 0.8 mg L ± 1 . Growth of mycorrhizal and nonmycorrhizal plants was poor in the medium not amended with P (Table 2).

Discussion The knowledge gained from the incubation study about the P sorption isotherm of the peat-based medium was critical in the identi® cation of a solution P concentration that was most conducive to the production of mycorrhizal L . leucocephala. The decline in solution P concentration observed in the medium during the incubation study and following plant growth in it are not unexpected. (Bolan 1991). However, the speed of the P reactions and the extent of the decline are known to be related to the speci® c physiochemical characteristics of the medium and the amount of P added (Bolan 1991; Barber 1995). The di€ erences in the P sorption isotherms produced by the method of Fox and Kamprath (1970) and by our incubation approach illustrate the need for tailoring procedures of P sorption isotherms to speci® c media. The incubation approach was more appropriate to the physiochemical characteristic of the peat-based medium tested than was the Fox and Kamprath method (1970). The incubation of the peat-soil medium as a suspension as prescribed by the latter approach could lead to anaerobic conditions due to the metabolism of peat components by microorganisms during incubation. Because protons are used up during anaerobic metabolism, the pH of the medium will increase, leading to increased availability of P. Moreover, a portion of the proton pool can be used in the reduction of iron oxide, resulting in the release of the P adsorbed on it (Singer and Munns 1991). These phenomena are probably responsible for the extraordinarily high

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solution P concentrations observed with the Fox and Kamprath approach, making the approach unsuited for our purpose. The range of solution P concentrations established in the peat-based medium by following the incubation approach enabled us to monitor the e€ ect of external P on mycorrhizal colonization in the medium and to test mycorrhizal response of L . leucocephala to P concentration in the medium. This approach is di€ erent from those employed in previous studies in which either a range of P concentrations was added without regard for P attained in solution (Graham and Timmer 1984; Pedersen et al. 1991) or in which two arbitrary levels of equilibrium P concentrations were tested based on what the authors considered to be low or high applications (Biermann and Linderman 1983). While the presence of mycorrhizas is expected to lead to a greater depletion of P from the growth substrate due to increased P uptake, the di€ erences we observed between media supporting mycorrhizal and nonmycorrhizal plants at the termination of the growth period were more pronounced than previously reported (Habte and Manjunath 1987) (Table 1). This may be related to the lower P sorption capacity of the peat-based medium relative to soil-based growth substrates. The peat-based medium lacks appreciable amounts of P sorption sites (anion exchange capacity) and is dominated by a net negative charge of the organic matter at pH values favorable for plant growth (Bohn et al. 1985). These characteristics limit the medium’s ability to maintain a P concentration in solution and to replenish P following depletion due to plant uptake. Thus, when AMF colonization substantially increased plant P uptake relative to nonmycorrhizal plants (Table 3), the medium was not able to replenish P at a rate su cient enough to o€ set removal by plants. This resulted in a much lower concentration of solution P concentration in the medium supporting mycorrhizal plants (Table 1). P status of the test medium signi® cantly in¯ uenced the extent of AMF colonization of L . leucocephala. AMF colonization decreased linearly with increasing solution P concentration (Figure 3), and colonization was highest at target solution P concentrations of 0.2 mg L ± 1 and 0.4 mg L ± 1 , colonization values at these two solution P concentrations not being signi® cantly di€ erent from each other (Table 2). The lack of AMF colonization in the plants growing in the medium which did not receive P suggests that the inherent solution P concentration of the medium was below the threshold limit for AMF colonization. At such low concentration of P, plant roots may not develop su ciently for mycorrhizal colonization (Habte and Fox 1993; Johnson 1976). Moreover, the host and the endophyte may compete for scarce P. The inverse relationship between AMF colonization and medium P concentration observed in this study is similar to those observed in soil-based media (Habte and Manjunath 1987; Sylvia and Neal 1990; Khaliq and Sanders 1997; Mendoza and Pagani 1997). However, this e€ ect has not been as clearly demonstrated in peatbased media. Pedersen and others (1991) did not ® nd a signi® cant decrease in AMF colonization with applied P. Both Johnson and Hummel (1986) and Biermann and Linderman (1983) found decreases in mycorrhizal infection with increasing proportions of peat, but in neither study was P recognized as the cause of depressed colonization. However, Graham and Timmer (1984) noted a sharp decrease in AMF colonization when double-acid extractable P increased above 8 mg P g ± 1 . Mycorrhizal plants maintained higher pinnule P concentration than nonmycorrhizal plants over the range of target solution P concentrations tested. Solution P concentrations which were favorable to AM fungal colonization were also most conducive to plant P uptake, suggesting a very strong correlation between AM fungal colonization and AM symbiotic e€ ectiveness. Based on these observations, we conclude that the optimal solution P concentration for mycorrhization in media with similar P sorption characteristics as our test medium is 0.2 to 0.4 mg L ± 1 . However, when one considers AMF colonization and growth, the optimal solution

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concentration for the development of mycorrhizal seedlings is 0.2 mg L ± 1 . This solution P concentration is 10 times that observed in soil-based medium (Habte and Manjunath 1987, 1991). In fact, the concentration of P we considered optimal in peat-based medium is known to decrease AM dependency of L . leucocephala in soil (Habte and Manjunath 1991). In addition, the inherent solution P concentration of the peat-based medium was insu cient for mycorrhizal and nonmycorrhizal plant growth in this experiment, even though a similar concentration was shown to be more than adequate for the growth of mycorrhizal L . leucocephala in soil (Habte and Manjunath 1987, 1991). These results suggests that in systems characterized by low P bu€ er capacity as in the peat-based medium tested, higher solution P concentrations may be required to compensate for the inability of the systems to maintain solution P concentration against removal by plants. Woodru€ and Kamprath (1965) found that soils with a low adsorption capacity (low P bu€ er capacity) needed a greater saturation of the P adsorption sites and a higher equilibrium solution concentration for maximum growth because the low P sorption capacity limited the soil’s capacity to continuously renew the soil solution P. This is consistent with the conclusion of Fox and Kamprath (1970) that in low P sorption systems, the concentration of P in the solution alone may not be the overriding factor of plant P nutrition. Mycorrhizal plants reached yields comparable to that of nonmycorrhizal plants at a target P concentration which is one-quarter than that necessary for mycorrhiza-free growth. Habte and Manjunath (1987) interacted L . leucocephala and G. fasciculatum in a fumigated soil at a range of solution P concentrations and noted that the concentration of P required to grow mycorrhizal plants was approximatel y one-twenty-seventh of that required to grow nonmycorrhizal plants of similar size. This di€ erence indicates that the external P requirement of mycorrhizal and nonmycorrhizal L . leucocephala may vary depending on the type of growth medium. This variation, we believe, re¯ ects the di€ erences in the P bu€ er capacity of the media.

Conclusion The results of the current investigation reiterate the observation that solution P concentrations in growth media can play an important role in regulating the AM symbiosis (Manjunath and Habte 1990; Habte and Manjunath 1987). However, external P requirements for raising mycorrhizal seedlings may di€ er depending on the characteristics of growth media. Identifying optimum P concentrations for mycorrhizal development can be accomplished by establishing a range of target solution P concentration appropriate for the particular medium. The most e€ ective way of doing this is by creating a P sorption isotherm and establishing desired solution P concentrations using the P sorption isotherm as a guide.

References Barber, S. A. 1995. Soil nutrient bioavailability: A mechanistic approach. Wiley, New York. Bethlenfalvay, G. J. 1992. Mycorrhizae and crop productivity. In Mycorrhizae in sustainable agriculture, edited by G. J. Bethlenfalvay and R. G. Linderman, 1± 27. American Society of Agronomy, Madison, Wisconsin. Biermann, B., and R. G. Linderman. 1983. E€ ect of container plant growth medium and fertilizer phosphorus on phosphorus on establishment and host growth response to vesicular-arbuscular mycorrhizae. Journal of American Society of Horticultural Science 108:962± 971. Bohn, H. L., B. L. McNeal, and G. A. O’Connor. 1985. Soil chemistry. Wiley & Sons, New York. Bolan, N. S. 1991. A critical review on the role of mycorrhizal fungi in the uptake of phosphorus by plants. Plant and Soil 134:189± 207.

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Brady, N. C., and R. R. Weil. 1999. T he nature and properties of soils. Prentice Hall, Upper Saddle River, New Jersey. Fox, R. L., and E. J. Kamprath. 1970. Phosphate sorption isotherms for evaluating the phosphate requirements of soils. Soil Science Society of America Proceedings 34:902± 907. Giovannetti, M., and B. Mosse. 1980. An evaluation of techniques for measuring vesiculararbuscular mycorrhizal infection in roots. New Phytologist 84:489± 500. Graham, J. H., and L.W. Timmer. 1984. Vesicular-arbuscular mycorrhizal development and growth response of rough lemon in soil and soilless media: E€ ect of phosphorus source. Journal of American Society of Horticultural Science 109:118± 121. Habte, M. 1992. Usefulness of the pinnule technique in mycorrhizal research. Methods in Microbiology 24:323± 337. Habte, M., and R. L. Fox. 1993. E€ ectiveness of VAM fungi in nonsterile soils before and after optimization of P in soil solution. Plant and Soil 151:219± 226. Habte, M., and A. Manjunath. 1987. Soil solution phosphorus status and mycorrhizal dependency in L eucaena leucocephala. Applied and Environmental Microbiology 53:797± 801. Habte, M., and A. Manjunath. 1991. Categories of vesicular-arbuscular mycorrhizal dependency of host species. Mycorrhiza 1:3± 12. Habte, M., and M. Soedarjo. 1995. Limitation of vesicular-arbuscular mycorrhizal activity in L eucaena leucocephala by Ca insu ciency in an acid Mn-rich oxisol. Mycorrhiza 5:387± 394. Habte, M., Y. C. Zhang, and D. P. Schmitt. 1999. E€ ectiveness of Glomus species in protecting white clover against nematode damage. Canadian Journal of Botany 77:135± 139. Hoagland, D. R., and D. I. Arnon, 1950. T he water-culture method for growing plants without soil. California Agricultural Experiment Station Circular 347. Berkeley, California. Johnson, P. N. 1976. E€ ect of soil phosphorus level and shade on plant growth and mycorrhizas. New Zealand Journal of Botany 14:333± 340. Johnson, C. R., and R. L. Hummel. 1986. In¯ uence of media on endomycorrhizal infection and growth response of Severinia buxifolia. Plant and Soil 93:35± 42. Khaliq, A., and F. E. Sanders. 1997. E€ ects of phosphorus application and vesicular arbuscular mycorrhizal inoculation on the growth and phosphorus nutrition of maize. Journal of Plant Nutrition 20:1607± 1616. Kormanik, P. P., W. C. Bryan, and R. C. Shultz. 1980. Procedure and equipment for staining large numbers of plant root samples for endomycorrhizal assay. Canadian Journal of Microbiology 26:536± 538. Manjunath, A., and M. Habte. 1990. Establishment of soil solution P levels for studies involving vesicular-arbuscular mycorrhizal symbiosis. Communications in Soil Science and Plant Analysis 21:557± 566. Mendoza, R. E., and E. A. Pagani. 1997. In¯ uence of phosphorus nutrition on mycorrhizal growth response and morphology of mycorrhizae in L otus tenuis. Journal of Plant Nutrition 20:625± 639. Murphy, J., and J. P. Riley. 1962. A modi® ed single solution method for the determination of phosphate in natural waters. Analytica Chimica Acta 27:31± 36. Onguene, N. A., and M. Habte. 1995. Nitrogen and phosphorus requirements for raising mycorrhizal seedlings of L eucaena leucocephala in containers. Mycorrhiza 5:347± 356. Pedersen, C. T., G. R. Sa® r, S. Parent, and M. Caron. 1991. Growth of asparagus in a commercial peat mix containing vesicular-arbuscular mycorrhizal (VAM) fungi and the e€ ects of applied phosphorus. Plant and Soil 135:75± 82. Schreiner, R. P., and G. J. Bethlenfalvay. 1995. Mycorrhizal interactions in sustainable agriculture. Critical Reviews in Biotechnology 15:271± 285. Singer, M. J., and D. N. Munns. 1991. Soil, an introduction. MacMillan, New York. Sylvia, D. M., and L. H. Neal. 1990. Nitrogen a€ ects the phosphorus response of VA mycorrhiza. New Phytologist 115:303± 310. Woodru€ , J. R. and E. J. Kamprath. 1965. Phosphorus absorption maximum as measured by the Langmuir isotherm and its relationship to phosphorus availability. Soil Science Society of America Proceedings 29:148± 150.