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P absorbed from soil by mycorrhizal red clover plants as affected by soluble P fertilization
Soil Bid. Biockm. Vol. 20. NO. 1. pp. 61-67. 1988 Printed in Great Britain. All rights reserved
Copyright 0
0038-07~7iaa~3.00 + 0.00 1988 Pergamon Journals Ltd
P ABSORBED FROM SOIL BY MYCORRHIZAL RED CLOVER PLANTS AS AFFECTED BY SOLUBLE P FERTILIZATION MARIA J. SAINZ and
JUSTOARINES
U.E.I. de Nutrici6n Vegetal y Fertilidad de Suelos, Instituto de Investigaciones Agrobiolbgicas de Galicia,
C.S.I.C.. Apartado 122, Santiago de Compostela, Spain (Accepted 25 May 1987) Summary-Three acid hill soils were used to study the effect of applied P on red clover growth and on mycorrhizal infection by native VAM endophytes, and the origin of P absorbed from soil by the plant. Plant growth increased and VAM infection decreased significantly with increasing P doses. The fractions of soil inorganic P extracted with 0.5 N NH, F and 0.1 N NaOH retained most of the P applied. Plants mainly utilized inorganic P of the soil fraction extracted with 0.5 N NH,F. but no absorption of P from the organic P-fractions was detected.
MATERIALSAND SWTHODS
INTRODUCTION One of the most important aspects of vesiculararbuscular mycorrhizal (VAM) studies is the interaction between VAM fungi and the phosphate applied to the soil to reach optimum crop productivity.
This interaction depends both on the infectivity and efficiency of the VAM endophytes and on the sorption characteristics of the soil, which affect the concentration of P in the soil solution and hence the requirement for fertilizer. As VAM fungi enhance P uptake by plants (Abbott and Robson, 1982) but high P fertilization decreases the extent of fungal infection (Bolan and Abbott, 1983), a compromise situation must exist where optimal benefits from mycorrhiza and fertilizer may be achieved. The study of this interaction is particularly important in practice where high inputs of P are usually needed to obtain good dry matter production. Of interest also is the source of soil P used by the plant. As stated by Chang and Jackson (1957), soil phosphate is bound to different surfaces, which contain Al, Fe, Ca or organic matter, depending on the nature of the soil. Organic-P plays an important role in P dynamics, particularly in soils with a high organic matter content. Bloom (1981) has found that Al-organic matter complexes strongly affect P adsorption in those soils, and Mugambi (1983) has also reported a close relationship between soil P sorption capacity and organic matter content. The use of organic-P by crops has been studied by comparing virgin and cultivated soils by fractionation techniques (Hedley et al., 1982; Tiessen et al., 1984; Sharpley and Smith, 1985), but no attempts have been made to relate changes in P-fractions obtained from cultivated and non-cultivated soils within an experimental trial. We attempted to establish adequate soil P levels for red clover growing in three acid hill soils with high organic matter contents. The effect of applied P on mycorrhizal infection by native VAM endophytes, the optimum mycorrhizal-fertilizer equilibrium, and the origin of the P absorbed from soil by the plant in terms of changes in soil P fractions (both inorganic and organic) were also studied. 61
Samples from the A horizon (0-20cm depth) of three hill soils (humic cambisols) were used. Chemical characteristics are summarized in Table I. Exchangeable Al was neutralized by liming as recommended by Kamprath (1970); as a result, pH (H,O) at the end of the experiment was 5.3, 5.3 and 5.5 for Mayor, Anton and Curra soils, respectively. At IS days after liming, samples of each soil were taken to determine: (i) P sorption isotherms, (ii) changes in inorganic-P fractions as affected by a large addition of phosphate, and (iii) P fertilizer doses to be used in the experiment. (i) P sorption isotherms were studied as described by Fox and Kamprath (1970). Samples of soil were incubated with the following doses of Ca(H,PO,),. Hz0 (pg P g-’ soil): 0, 100,200,400,600, 800, 1000, 1500, 2000 and 2500. (ii) Inorganic-P fractions were obtained by the method of Chang and Jackson (1957) as described by Arines and Alvarez (1981): soil was sequentially extracted with 1 N NH,CI, 0.5 N NHIF, 0.1 N NaOH, solid sodium dithionite and tribasic sodium citrate (DC), and 0.5 h’ H2S0,; finally, samples were calcinated and extracted again with 0.5 N HZS04. (iii) Samples of soil were incubated for 4 days with the following doses of KH,POb @g P g-’ soil); 0, 30, 60, 90, 120, 150 and 300 (I :2 soil: solution ratio). Then IOmM CaCI,-, Olsen-, Bray-2-, and Mehlichavailable P (Olsen et al., 1954; Bray and Kurtz, 1945; Mehlich, 1978) were measured (Table 2). The Bray-2 method was modified by using I :20 soil:solution ratio and 20 min of shaking. P fertilizer doses were established according to McLean et al. (1982). An additional high dose was also included for each soil to obtain a complete growth-response curve. The following P doses (pg P g-l soil) as KHIPO, were applied: 0 (PO), 125 (PI), 250 (P2). 500 (P3) and 1000 (P4) for Mayor and Curra soils, and 0 (PO), 50 (PI), 100 (PZ), 200 (P3) and 300 (P4) for Anton soil. There were 8 replicates per treatment, each sown with 10 seeds of Trifolium prutense L. (2 seeds in five equidistant positions) 4 days after P addition. The num-
>L~IA J. SAWZ and JLSTOARINES
her of seedtings was reduced to ii&-eafter the appearance of the first trifofiare leaf. Two P fertilization treatments (pg. P g-’ soil) as RHzPOI were also set up in non-cufttvated soil: 0 and 500 for Mayor and Curra soils, and 0 and 200 for Anton soil, with 4 replicates per treatment. The experiment was carried out in a glasshouse from May to October, the air temperature being 18-25/g-12’C, and pots containing 7OOg soil were randomly arranged. Soil moisture content m each pot was adjusted daily with distilled water by weighing. Ten ml of a nutrient solution lacking P were added every 2 weeks. The first harvest was carried out 3 months after sowing, using four repticates per treatment. Fresh weights of shoots and roots were jmrn~diat~iy recorded. Shoots were cut 2 cm above the soil surface and. after removing senescent plant material, dried to constant weight in an oven at 7O’C. After grinding, 100 mg of dried plant material were ashed in a muffle furnace at 450°C. P concentrations in hot 5 N NCI digests of the calcinatcd shoots were determined with sulphomolybdic reagent and ascorbic acid as reducing agent (Dick and Tabatabai. 1977). The remaining four pots were also cut and left for the second harvest. Roots were gently washed and cut in 2cm segments. Samples ta assess VAM infection were taken, cleared and stained according to Phillips and Hayman (1970). Percent VAM root cofonization was estimated by the gridfine intersect method. observing 200 intersection points per sample (Giovannetti and Mossc, 1980). A representative soil sample was taken from each pot to study inorganic and organic P fractions, using a procedure based on Hedley et al. (1982) and Tiessen et al. (1984) (Fig. 1). Data from soil P fractionation were used to cab late P absorbed by plants, as follows: for each fraction, values obtained in non-cultivated soil were used as a reference, i.e. X0 = P extracted at PO dose and X3 = P extracted at P3 dose; X3 - X0 indicated the amount of apptied P retained in the fraction when P3 dose was added. In this way, the theoreticaf percent P retention (Z‘S) coufd be estimated for each fraction, Assuming that percent P d~strjbution does not change with P dose, the theoreticat P quantity (Wd) that should be in each fraction was calculated for each dose (Pd): Wd = Z% x Pd. P absorbed by plants was obtained determining the difference between the theoretical amount of P in non-cultivated soil (Wd) and the one measured in cultivated pots (Yd) for each P dose: P absorbed = Wd - I’d. The remaining pots (4 replicates per treatment) were harvested 5~ months after sowing. Fresh weights of shoots and roots, dry weights of shoots and percent VAM infection were studied. Data were subjected to a two-way analysis of variance (ANOVA test); ieast significant differences were calculated by an F-test.
RESULTS
More P was adsorbed by the Mayor soil than by the other two, which both showed similar adsorption curves (Fig. 2); nearly 2000 mg P kg-’ soil were needed in the former soil to achieve I mg P kg- ’ soit
P absorbed
by myeorrhizai red clover plants
Table 2. 10 mxt CaCl,-, Olsen-, Bray-t-, and Mehli~-a~~iable i-sing doses of KH,PO, Soil
5xtrsctant
Mayor
IOmt4 CaCI,
OISCII Anton
Curra
Bray-2 Mchlich IO mxt CaCI, Olxn Bray-2 Mehlicb IO mxt CaCI, OlKn Bray-2 Mehlich
0
63
P aRer soil ~ncu~tion witb
Incubation doses (fig P g-’ soil) 30 60 90 120 1%
0 II 68 5 0 68 213 18 0 6 30 2
remaining in solution compared to loo0 mg in the latter two. P fertilizer was mainly retained in the fraction of inorganic P extracted with 0.5 N NHIF from the Mayor and Anton soils (56 and 58%, respectively). In the Curra soil, the inorganic P fraction extracted with
0
0
0
IS 73 5 0 71 253 28 0 It 44 4
20 88
29 96 12 0
I:: 17 0
251: 40 0 29 51 19
2:: 49 0 53 58 25
It 76 288 33 0 17 34 12
0
300
0
0 46 130 16 0 96 339 55 0 SS 77 35
70 211 30 0 138 437 92 0 88 135 77
0.1 N NaOH retained most of the P applied (64%) (Fig. 3). In all three soils, plant growth increased markedly with increasing P doses (Fig. 4). Addition of P2 dose in Mayor soil and Pl dose in Anton and Curra soils resulted in dry matter productions similar to the
SOIL Duplicate samples, 1 g of soil (co.5 mm) in centrifuge tubes with screw-top. Add 5Omi of 1Omt.r Cat&. Shake 1 h. Centrifuge and filter. Analyze supernatant .._.........,...,......._......... P in soil solution (inorganic P)
I SOIL Add 50 ml of 0.5 N NH,F.
pH 5. Shake 1 h
Centrifuge and filter. Analyze supernatant .......... ................... ........ ......................... NH,F-P
(Inorganic
P)
I Wash with 25ml of saturated NaCl
Take an aliquot of 5ml. Cal&ate and recover the ashes with 1 N &SO,. Centrifuge and filter. Analyze ........................ NH,F-P
(Total P)
SOIL Add 50 ml of 0.1 N NaOH. Shake 17 h. Proceed as before. In the supernatant ._...._.__..........,,.............................................. NaOH-P
(Inorganic P)
Proceed as before. Analyze . .................. ..... NaOH-P
(Total P)
Add 50ml of 0.1 N NaOH. Sonicate for 5min. Shake 17 h. Proceed as before. In the supernatant . . . . . . .. ... NaOH U.S.-P (Inorganic P) Wash as before
Take an aliquot of 5ml and proceed as before. Analyze ................ NaOH U.S.-P (Total P)
I SOIL Add 5Oml of 1 N &SO,. Shake 1 h. Proceed as before. Analyze supernatant ...... H,SO,-P (inorganic P)
I SOIL Transfer to a capsule with distilled water. Evaporate to dryness. Ash 2 h at 550X Collect residue with 1 N HsSO,. Shake 1 h Centrifuge and filter. Analyze supernatant .........._......................................
t............. HsSO,-P
(Organic P)
Organic-P is calculated as the difference between Total P and Inorganic P in NH, F and NaOH extracts.
Fig. I. Scheme of the method followed to study inorganic and organic P of the soil.
MARIA J. SAINZ
64
”
”
”
J
1
01234567%
Fig. 2. Phosphate adsorption isotherms for Mayor (e). Anton (8) and Curra (A) soils after equilibration for 6 days.
‘*I
la1
2w
0-
NH&i W&f NaOH DC I+&, H2G Extractantsoluhons
Fig. 3. Mean values of inorganic P fractions extracted from (a) Mayor, (b) Anton and (c) Curra soils with no P added (0) and after incubation with 500 (a and c) or 300 (b) pg P g-’ soil as KH,PO, (($1).
and
JUTO
maxima. Phosphorus concentrations in the shoots also increased with applied phosphate. the highest percentage being in plants grown in Curra soil when IOOOmg P kg-’ soil were added. This concentration was, however. associated with shoot dry weight and P-uptake values lower than with 500 mg P (Fig. 4). P concentrations for the P2 treatment in Mayor soil and Pl in Anton and Curra soils were about 0.20%. this percentage corresponding with a strong decrease in VAM infection (Fig. 5). P-uptake by plants was similar in all three soils at PO and Pi levels. but plants grown in Anton soil took up significantly less phosphate than in the other two at P2 and P3 levels (Fig. 4). The P3 dose led in all cases to P-uptake values equal to the maxima. Statistical significance of plant dry weight and P nutrition data are shown in Table 3. VAM root infection was greatly diminished by the lowest P dose (PI) in each soil, being about 20% or less when soils were fertilized with more than 200 mg P kg-’ soil (Fig. 6). Most VAM root infection was found when no P was added, although VAM fungi in the Mayor soil infected only 50% of the root length. Infections by both fine and coarse endophytes were observed in roots grown in each soil and fragments of roots simultaneously colonized by both types of endophytes were noted. At the second harvest, fresh and dry weights of plant shoots were lower than in the first one (data not shown) and showed a high variability. This lower yield may have resulted from a depletion of soil nutrients, including P, as suggested by Lim and Cole (1984). Nevertheless, the same trends in dry matter production were observed as in the first cut, the Mayor soil reaching the highest yields. Root fresh weights were higher than in the first harvest, particularly at the highest doses of P applied (Fig. 7). The analysis of percent VAM infection showed that VAM root colonization decreased with higher P doses, but a significant increase in infection was observed compared to the first harvest (Fig. 6). In ail three soils, plants mainly absorbed inorganic P corresponding to that extracted with 0.5 N NE&F (Fig. 8). The amount of P utilized from this fraction was higher in the Mayor and Curra soils, where the absorption increased with increased P addition. Inorganic P extracted with 0.1 N NaOH was also used, although to a lesser extent. No absorption of P by plants from the organic P-fractions was noted (Fig. 8), but the methodology followed may not detect changes in the organic P in a short-time period.
. __ 8
a
8
0
*
8
0
lO9%765432100 P-uptake(mglpotl
8
3
3
a
AWES
. 1
I
01
0.2
03
a4
0.5 % P
Fig. 4. Relationship between dry wt and %P and P-uptake of the shoots in Mayor (0). Anton (m) and Curra (A) soils.
P absorbed by mycorrhizal red clover plants
,
t 05 %
OL
t,
03
02
,
I
,
*
I
,
,
6.5
,
I
(
001236567090
01
P
P-uptake &t/pot)
Fig. 5. Relatjonship between %VAM
infection and %P and P-uptake of the shoots in Mayor (e)b Anton (8) and Curra (A) soils.
Table 3. Statisticalsignificance of red clovergrowthand P nutritionparametersin the three soilsas relatedto increasingP doses.First harvest Significance Soil Shoot dry WI (8 pot-‘1 Shoot P% Shoot P-uptake (mg pot-‘)
Phosphorus P
P < 0.01 P < 0.01
I.20 0.09
P co.01
P < 0.01
P < 0.01
2.99
increasing P addition enhanced plant growth at both harvests in all three soils studied. The highest P
dose did not result in a significantly increased yield respect to the P3 dose, thus confirming the predictions for P fertilizer needs by the McLean er al. method (1982).
r
LSD Interaction
P
DISCUSSfON
100
Interaction
(al
Differences in plant growth with increasing P supply were probably related to P-retention characteristics of the three soils, but a major role of the native VAM fungal populations in improving P-uptake by colonized roots, particularly at the lowest phosphate treatments, can not be discarded. VAM fungi have been shown to increase crop productivity and Puptake when colonizing roots grown in P-deficient soils or when low to moderate P doses are applied (Hayman, 1983). The mycorrhizal effect can not be established in our experiment, but the results on VAM infection and plant growth make us think that the symbiosis may have been effective at the lowest P doses or when no P was added, particularly in the Mayor soil, which reached the highest yields although showing the highest P retention properties (Fig. 2). Nevertheless, yields obtained with the highest P doses may have been a direct effect of P addition
20 400 600 800 1040 Phosphate appliedhg P/kg so11 I
0 Fig. 6. Effect of P addition on %VAM infection for Mayor (0). Anton (m) and Curt-a (A) soils. (a) 1st harvest, (b) 2nd harvest. 4 indicates that the increment in %VAM infection between the 1st and the 2nd harvest is significantly different at the 5% level.
200
LOO 600
800
1000
Phosphate applied(mgP/ Kg so11 I Fig. 7. Effect of P addition on root fresh wt for Mayor (a).
Anton (m) and Curra (A) soils. 0. n , A 1st harvest. 0, CJ, A 2nd harvest.
MARIA
66
J.
SAWZ
and JUSTO AR~XE.V
P-fertilizers are quickly adsorbed onto the most active surfaces of acid soils, bearing di- or trivalent LOO (or ions (Al, Fe, Ca) (Russell, 1973): this is confirmed in * our results were applied P was mainly retained in the 300 ,f ,. inorganic P-fractions extracted with NH,F (P bound F w :. to Al) and NaOH (P bound to Fe). These fractions are the principal sources of orthophosphate ions to the soil solution, as the plant and VAM fungi deplete available P. Our study of P absorption by plants agrees with this, since inorganic P extracted with NH,F was the fraction mainly utilized (Fig. 8). Vesicular-arbuscular mycorrhiza may have greatly contributed to the absorption of this fraction when the lowest P doses were applied, as Ross and Gilliam (1973) found that this fraction was better used by mycorrhizal than non-mycorrhizal plants. It is known g 200 that both fungal hyphae and roots take up phosphate from the labile pool of the soil (Mosse ef al., 1973), % 100 but Cress et nl. (1979) reported that fungal hyphae have a greater affinity than roots to phosphate. 0 Differences in P-affinity between VAM species may Pl P2 P3 Pl P2 P3 Pl P2 P3 Pl P2 P3 be related to their ability to take up the phosphate reIeased from NH, F- and NaOH-extracted inorganic Phosphate apptted P-fractions, particularly in soils with high PFig. 8. Inorganic P (P,) and organic P (P,,) utilized by plants retention. The fact that no utilization of organic P by from the soil fractions extracted with 0.5 N NH,F and 0. I N plants was detected further supports that only labile NaOH. (a) Mayor, (b) Anton and (c) Curra soils. 88 P P is readily available both to plant and VAM fungi, utilized. jr indicates that the utilization is significant at the at least in a short term. 5% level. The higher P-adsorption characteristics of the Mayor soil may have caused a greater dependence of red clover on mycorrhiza, and the presence of a rather than of mycorrhiza. Parfitt ef al. (1982) found that mycorrhiza had no effect at high levels of added highly effective VAM population may account for the P and Rangeley et al. (1982) observed that ~~~~~~~rn enhanced plant growth compared to the other two repens only responded to VAM infection when P soils. concentrations in the shoots were below [email protected]% Acknowledgemenrs-We thank Dr D. S. Hayman for This value was associated in our work .with VAM critical and grammatical revision of thr manuscript. colonization of IO-30% of the root, probably too low to account for the growth effect recorded in the first REFERENCES harvest. The decrease in VAM root infection on adding P Abbott L. K. and Robson A. D. (1982) The role of vesicular-arbuscular mycorrhizal fungi in agriculture and fertilizer has been widely reported in mycorrhizal the selection of fungi for inoculation. Ausfruliun Journal studies, this response being characterized by a maxiof Agticulrurat R&&h 33, 3891108. mum infection value when no P is applied and a Arines J. and Alvarez M. T. (19811 Contribucibn al estudio progressive decrease in VAM colonization as P applidel f&for0 en suelos icidos. i. Caracteristicas de adcation increases (Gianinazzi-Pearson, 1985). The ineorcibn y fracciones de P en una tierra parda de1 noroeste ternal P concentration of the plant, as a result of a de Esparia. Turrialha 31, 217-226. high P level in the soil, has been claimed as the factor Bloom P. R. ((981) Phosphorus adsorption by an aluminium-peat complex. Soil Science Sociery of’cimrrica controlling mycorrhizal colonization (Menge er al., Journal 45, 267-212. 1978). In this respect, at the first harvest, we found Bolan N. S. and Abbott L. K. (1983) Seasonal variation in inhibitory P concentration values in the plant for infectivity of vesicular-arbuscular mycorrhizal fungi in VAM infection to be 0.20425% and over, correrelation to plant response to applied phosphorus. Austrasponding to the addition of more than 2OOpg P g-’ lian Journuf of Sod Research 21. 208-210. to the soil. Therefore, this amount of soluble phosBray R. H. and*Kurtz 1. T. (1945) Determination of total phate marks the limit for P fertilization in the soils inorganic and available forms of phosphorus in soils. Soil studied if optimal benefits from P inputs and VAM Science 59, 39-45. infection are to be obtained. Chang S. C. and Jackson M. L. (1957) Fractionation of soil phosphorus. Soil Science 84, 133-11-1. P concentration of the plant was probably reCress W. A., Throneberry G. 0. and Lindsay D. L. (1979) sponsible for the increase in VAM colonization from Kinetics of phosphorus adsorption by mycorrhizal and the first to the second harvest at some P doses, nonmycorrhizal tomato roots. Plant Physiology 64, particularly in Mayor and Anton soils (Fig. 6). 484-487. despite the fact that root growth was higher (Fig. 7) Day P. R. (1965) Particle fractionation and particle-size and might have induced a dilution effect in the analysis. In Merhodr of Soil Analysis (C. A. Black et al., infection. According to Menge et al. (1978). P conEds), pp. 545-566. American Society of Agronomy, centration of the plant should have been lower to Madison. allow the higher percentages of infection recorded at Dick W. A. and Tabatabai M. A. (1977) An alkaline oxidation method for determination of total phosphorus the second harvest. NH&F-P.
\a3H-P,
NHLF-?a
NoOH-P.
P absorbed by mycorrhizal red clover plants in soils. Soil Science Society of America Journal 41, 51 l-514. Fox R. L. and Kamprath E. J. (1970) Phosphate sorption isotherms for evaluating the phosphate requirements of soils. Soil Science Society of America Proceedings 34, 902-907.
Gianinazzi-Pearson V. (1985) Mycorrhizal effectiveness in phosphate nutrition: how, when and where? In Proceedings of the 6th North American Conference on Mycorrhizae (R. Molina. Ed.), pp. 15&154). Bend, Oregon.
Giovannetti M. and Mosse B. (1980) An evaluation of techniques for measuring vesicular-arbuscular mycorrhizal infection in roots. New Phytologisr 84.489-500. Hayman D. S. (1983) The physiology of vesiculararbuscular endomycorrhizal symbiosis. Canadian Journal of Bofany 61, 944-963.
Hedley M. J., Stewart J. W. B. and Chauhan B. S. (1982) Changes in inorganic and organic soil phosphorus fractions induced by cultivation practices and by laboratory incubations. Soil Science Society of America Journal 46, 970-976.
Kamprath E. J. (1970) Exchangeable aluminum as criterion for liming leached mineral soils. Soil Science Society of America Proceedings
34, 252-254.
Lim L. L. and Cole A. L. J. (1984) Growth response of white clover to vesicular-arbuscular mycorrhizal infection with different levels of applied phosphorus. New Zealand Journal of Agricultural- Research 2;. 587-592.
McLean E. 0. (1965) Aluminium. In Merhoa!s of Soil Analysis (C. A.‘Black et al., Eds). pp. 978997. American Society of Agronomy, Madison. McLean E. 0.. Oloya T. 0. and Mostaghimi S. (1982) Improved corrective fertilizer recommendations based on a two step alternative usage of soil tests. I. Recovery of soil equilibrated phosphorus. Soil Science Sociery of America Journal 46, 1193-l 197.
Mehlich A. (1978) New extractant for soil test evaluation of phosphorus, potassium, magnesium, calcium, sodium, manganese and zinc. Soil Science and Plum Analysis 9, 477492.
Menge J. A., Steirle D., Bagyaraj D. J., Johnson E. L. V.
67
and Leonard R. T. (1978) Phosphorus concentrations in plants responsible for inhibition of mycorrhizal infections. New Phyrologist 80, 575-578. Mosse B.. Hayman D. S. and Arnold D. J. (1973) Plant growth responses to vesicular-arbuscular mycorrhiza. V. Phosphate uptake by three plant species from P-deficient soils labelled with ‘*P. New Phyrolonist 72. 809-815. Mugambi M. (1983) Soil organic mat& both a source and a sink for phosphorus in Kenya soils. Third fnremarional Congress on Phosphorus Compounds, October, pp. 587-59 I. IMPHOS, Brussels. Olsen S. R., Cole C. V.. Watanabe F. S. and Dean L. A. (1954) Estimation of available phosphorus in soils by extraction with sodium bicarbonate. U.S. Dept of Agriculture, Circular 939. ParIitt R. L., Hart P. B. S., Meyrick K. F. and Russell M. (1982) Response of ryegrass and white clover to phosphorus on an allophanic soil, Egmont black loam. New Zealand Journal of Agricultural
Research 25, 549-555.
Phillips J. M. and Hayman D. S. (1970) Improved procedures for clearing roots and staining parasitic and vesicular-arbuscular mycorrhizal fungi for rapid assessment of infection. Transactions of the Brirish Mycological Sociefy 55, I 59- I6 I. Rangeley A., Daft ht. J. and Newbould A. (1982) The inoculation of white clover with mycorrhizal fungi in unsterile hill soils New Phyfologist 92, 89-102. Ross 1. and Gilliam J. W. (1973) Effect of Endogone mycorrhiza on phosphorus uptake by soybeans from inorganic phosphates. Soil Science Society of America Proceedings
87, 237-239.
Russell E. W. (1973) The sources of plant nutrients in the soil: phosphate. In Soil Conditions and Plum Growrh, 10th ed. pp. 555603. Longman, London. Sharpley A. N. and Smith S. J. (1985) Fractionation of inorganic and organic phosphorus in virgin and cultivated soils. Soil Science Society of America Journal 49, 127-130.
Tiessen H., Stewart J. W. B. and Cole C. V. (1984) Pathways of transformations in soils of different pedogenesis. Soil Science Sociery of America Journal 48, 853-858.
Copyright 0
0038-07~7iaa~3.00 + 0.00 1988 Pergamon Journals Ltd
P ABSORBED FROM SOIL BY MYCORRHIZAL RED CLOVER PLANTS AS AFFECTED BY SOLUBLE P FERTILIZATION MARIA J. SAINZ and
JUSTOARINES
U.E.I. de Nutrici6n Vegetal y Fertilidad de Suelos, Instituto de Investigaciones Agrobiolbgicas de Galicia,
C.S.I.C.. Apartado 122, Santiago de Compostela, Spain (Accepted 25 May 1987) Summary-Three acid hill soils were used to study the effect of applied P on red clover growth and on mycorrhizal infection by native VAM endophytes, and the origin of P absorbed from soil by the plant. Plant growth increased and VAM infection decreased significantly with increasing P doses. The fractions of soil inorganic P extracted with 0.5 N NH, F and 0.1 N NaOH retained most of the P applied. Plants mainly utilized inorganic P of the soil fraction extracted with 0.5 N NH,F. but no absorption of P from the organic P-fractions was detected.
MATERIALSAND SWTHODS
INTRODUCTION One of the most important aspects of vesiculararbuscular mycorrhizal (VAM) studies is the interaction between VAM fungi and the phosphate applied to the soil to reach optimum crop productivity.
This interaction depends both on the infectivity and efficiency of the VAM endophytes and on the sorption characteristics of the soil, which affect the concentration of P in the soil solution and hence the requirement for fertilizer. As VAM fungi enhance P uptake by plants (Abbott and Robson, 1982) but high P fertilization decreases the extent of fungal infection (Bolan and Abbott, 1983), a compromise situation must exist where optimal benefits from mycorrhiza and fertilizer may be achieved. The study of this interaction is particularly important in practice where high inputs of P are usually needed to obtain good dry matter production. Of interest also is the source of soil P used by the plant. As stated by Chang and Jackson (1957), soil phosphate is bound to different surfaces, which contain Al, Fe, Ca or organic matter, depending on the nature of the soil. Organic-P plays an important role in P dynamics, particularly in soils with a high organic matter content. Bloom (1981) has found that Al-organic matter complexes strongly affect P adsorption in those soils, and Mugambi (1983) has also reported a close relationship between soil P sorption capacity and organic matter content. The use of organic-P by crops has been studied by comparing virgin and cultivated soils by fractionation techniques (Hedley et al., 1982; Tiessen et al., 1984; Sharpley and Smith, 1985), but no attempts have been made to relate changes in P-fractions obtained from cultivated and non-cultivated soils within an experimental trial. We attempted to establish adequate soil P levels for red clover growing in three acid hill soils with high organic matter contents. The effect of applied P on mycorrhizal infection by native VAM endophytes, the optimum mycorrhizal-fertilizer equilibrium, and the origin of the P absorbed from soil by the plant in terms of changes in soil P fractions (both inorganic and organic) were also studied. 61
Samples from the A horizon (0-20cm depth) of three hill soils (humic cambisols) were used. Chemical characteristics are summarized in Table I. Exchangeable Al was neutralized by liming as recommended by Kamprath (1970); as a result, pH (H,O) at the end of the experiment was 5.3, 5.3 and 5.5 for Mayor, Anton and Curra soils, respectively. At IS days after liming, samples of each soil were taken to determine: (i) P sorption isotherms, (ii) changes in inorganic-P fractions as affected by a large addition of phosphate, and (iii) P fertilizer doses to be used in the experiment. (i) P sorption isotherms were studied as described by Fox and Kamprath (1970). Samples of soil were incubated with the following doses of Ca(H,PO,),. Hz0 (pg P g-’ soil): 0, 100,200,400,600, 800, 1000, 1500, 2000 and 2500. (ii) Inorganic-P fractions were obtained by the method of Chang and Jackson (1957) as described by Arines and Alvarez (1981): soil was sequentially extracted with 1 N NH,CI, 0.5 N NHIF, 0.1 N NaOH, solid sodium dithionite and tribasic sodium citrate (DC), and 0.5 h’ H2S0,; finally, samples were calcinated and extracted again with 0.5 N HZS04. (iii) Samples of soil were incubated for 4 days with the following doses of KH,POb @g P g-’ soil); 0, 30, 60, 90, 120, 150 and 300 (I :2 soil: solution ratio). Then IOmM CaCI,-, Olsen-, Bray-2-, and Mehlichavailable P (Olsen et al., 1954; Bray and Kurtz, 1945; Mehlich, 1978) were measured (Table 2). The Bray-2 method was modified by using I :20 soil:solution ratio and 20 min of shaking. P fertilizer doses were established according to McLean et al. (1982). An additional high dose was also included for each soil to obtain a complete growth-response curve. The following P doses (pg P g-l soil) as KHIPO, were applied: 0 (PO), 125 (PI), 250 (P2). 500 (P3) and 1000 (P4) for Mayor and Curra soils, and 0 (PO), 50 (PI), 100 (PZ), 200 (P3) and 300 (P4) for Anton soil. There were 8 replicates per treatment, each sown with 10 seeds of Trifolium prutense L. (2 seeds in five equidistant positions) 4 days after P addition. The num-
>L~IA J. SAWZ and JLSTOARINES
her of seedtings was reduced to ii&-eafter the appearance of the first trifofiare leaf. Two P fertilization treatments (pg. P g-’ soil) as RHzPOI were also set up in non-cufttvated soil: 0 and 500 for Mayor and Curra soils, and 0 and 200 for Anton soil, with 4 replicates per treatment. The experiment was carried out in a glasshouse from May to October, the air temperature being 18-25/g-12’C, and pots containing 7OOg soil were randomly arranged. Soil moisture content m each pot was adjusted daily with distilled water by weighing. Ten ml of a nutrient solution lacking P were added every 2 weeks. The first harvest was carried out 3 months after sowing, using four repticates per treatment. Fresh weights of shoots and roots were jmrn~diat~iy recorded. Shoots were cut 2 cm above the soil surface and. after removing senescent plant material, dried to constant weight in an oven at 7O’C. After grinding, 100 mg of dried plant material were ashed in a muffle furnace at 450°C. P concentrations in hot 5 N NCI digests of the calcinatcd shoots were determined with sulphomolybdic reagent and ascorbic acid as reducing agent (Dick and Tabatabai. 1977). The remaining four pots were also cut and left for the second harvest. Roots were gently washed and cut in 2cm segments. Samples ta assess VAM infection were taken, cleared and stained according to Phillips and Hayman (1970). Percent VAM root cofonization was estimated by the gridfine intersect method. observing 200 intersection points per sample (Giovannetti and Mossc, 1980). A representative soil sample was taken from each pot to study inorganic and organic P fractions, using a procedure based on Hedley et al. (1982) and Tiessen et al. (1984) (Fig. 1). Data from soil P fractionation were used to cab late P absorbed by plants, as follows: for each fraction, values obtained in non-cultivated soil were used as a reference, i.e. X0 = P extracted at PO dose and X3 = P extracted at P3 dose; X3 - X0 indicated the amount of apptied P retained in the fraction when P3 dose was added. In this way, the theoreticaf percent P retention (Z‘S) coufd be estimated for each fraction, Assuming that percent P d~strjbution does not change with P dose, the theoreticat P quantity (Wd) that should be in each fraction was calculated for each dose (Pd): Wd = Z% x Pd. P absorbed by plants was obtained determining the difference between the theoretical amount of P in non-cultivated soil (Wd) and the one measured in cultivated pots (Yd) for each P dose: P absorbed = Wd - I’d. The remaining pots (4 replicates per treatment) were harvested 5~ months after sowing. Fresh weights of shoots and roots, dry weights of shoots and percent VAM infection were studied. Data were subjected to a two-way analysis of variance (ANOVA test); ieast significant differences were calculated by an F-test.
RESULTS
More P was adsorbed by the Mayor soil than by the other two, which both showed similar adsorption curves (Fig. 2); nearly 2000 mg P kg-’ soil were needed in the former soil to achieve I mg P kg- ’ soit
P absorbed
by myeorrhizai red clover plants
Table 2. 10 mxt CaCl,-, Olsen-, Bray-t-, and Mehli~-a~~iable i-sing doses of KH,PO, Soil
5xtrsctant
Mayor
IOmt4 CaCI,
OISCII Anton
Curra
Bray-2 Mchlich IO mxt CaCI, Olxn Bray-2 Mehlicb IO mxt CaCI, OlKn Bray-2 Mehlich
0
63
P aRer soil ~ncu~tion witb
Incubation doses (fig P g-’ soil) 30 60 90 120 1%
0 II 68 5 0 68 213 18 0 6 30 2
remaining in solution compared to loo0 mg in the latter two. P fertilizer was mainly retained in the fraction of inorganic P extracted with 0.5 N NHIF from the Mayor and Anton soils (56 and 58%, respectively). In the Curra soil, the inorganic P fraction extracted with
0
0
0
IS 73 5 0 71 253 28 0 It 44 4
20 88
29 96 12 0
I:: 17 0
251: 40 0 29 51 19
2:: 49 0 53 58 25
It 76 288 33 0 17 34 12
0
300
0
0 46 130 16 0 96 339 55 0 SS 77 35
70 211 30 0 138 437 92 0 88 135 77
0.1 N NaOH retained most of the P applied (64%) (Fig. 3). In all three soils, plant growth increased markedly with increasing P doses (Fig. 4). Addition of P2 dose in Mayor soil and Pl dose in Anton and Curra soils resulted in dry matter productions similar to the
SOIL Duplicate samples, 1 g of soil (co.5 mm) in centrifuge tubes with screw-top. Add 5Omi of 1Omt.r Cat&. Shake 1 h. Centrifuge and filter. Analyze supernatant .._.........,...,......._......... P in soil solution (inorganic P)
I SOIL Add 50 ml of 0.5 N NH,F.
pH 5. Shake 1 h
Centrifuge and filter. Analyze supernatant .......... ................... ........ ......................... NH,F-P
(Inorganic
P)
I Wash with 25ml of saturated NaCl
Take an aliquot of 5ml. Cal&ate and recover the ashes with 1 N &SO,. Centrifuge and filter. Analyze ........................ NH,F-P
(Total P)
SOIL Add 50 ml of 0.1 N NaOH. Shake 17 h. Proceed as before. In the supernatant ._...._.__..........,,.............................................. NaOH-P
(Inorganic P)
Proceed as before. Analyze . .................. ..... NaOH-P
(Total P)
Add 50ml of 0.1 N NaOH. Sonicate for 5min. Shake 17 h. Proceed as before. In the supernatant . . . . . . .. ... NaOH U.S.-P (Inorganic P) Wash as before
Take an aliquot of 5ml and proceed as before. Analyze ................ NaOH U.S.-P (Total P)
I SOIL Add 5Oml of 1 N &SO,. Shake 1 h. Proceed as before. Analyze supernatant ...... H,SO,-P (inorganic P)
I SOIL Transfer to a capsule with distilled water. Evaporate to dryness. Ash 2 h at 550X Collect residue with 1 N HsSO,. Shake 1 h Centrifuge and filter. Analyze supernatant .........._......................................
t............. HsSO,-P
(Organic P)
Organic-P is calculated as the difference between Total P and Inorganic P in NH, F and NaOH extracts.
Fig. I. Scheme of the method followed to study inorganic and organic P of the soil.
MARIA J. SAINZ
64
”
”
”
J
1
01234567%
Fig. 2. Phosphate adsorption isotherms for Mayor (e). Anton (8) and Curra (A) soils after equilibration for 6 days.
‘*I
la1
2w
0-
NH&i W&f NaOH DC I+&, H2G Extractantsoluhons
Fig. 3. Mean values of inorganic P fractions extracted from (a) Mayor, (b) Anton and (c) Curra soils with no P added (0) and after incubation with 500 (a and c) or 300 (b) pg P g-’ soil as KH,PO, (($1).
and
JUTO
maxima. Phosphorus concentrations in the shoots also increased with applied phosphate. the highest percentage being in plants grown in Curra soil when IOOOmg P kg-’ soil were added. This concentration was, however. associated with shoot dry weight and P-uptake values lower than with 500 mg P (Fig. 4). P concentrations for the P2 treatment in Mayor soil and Pl in Anton and Curra soils were about 0.20%. this percentage corresponding with a strong decrease in VAM infection (Fig. 5). P-uptake by plants was similar in all three soils at PO and Pi levels. but plants grown in Anton soil took up significantly less phosphate than in the other two at P2 and P3 levels (Fig. 4). The P3 dose led in all cases to P-uptake values equal to the maxima. Statistical significance of plant dry weight and P nutrition data are shown in Table 3. VAM root infection was greatly diminished by the lowest P dose (PI) in each soil, being about 20% or less when soils were fertilized with more than 200 mg P kg-’ soil (Fig. 6). Most VAM root infection was found when no P was added, although VAM fungi in the Mayor soil infected only 50% of the root length. Infections by both fine and coarse endophytes were observed in roots grown in each soil and fragments of roots simultaneously colonized by both types of endophytes were noted. At the second harvest, fresh and dry weights of plant shoots were lower than in the first one (data not shown) and showed a high variability. This lower yield may have resulted from a depletion of soil nutrients, including P, as suggested by Lim and Cole (1984). Nevertheless, the same trends in dry matter production were observed as in the first cut, the Mayor soil reaching the highest yields. Root fresh weights were higher than in the first harvest, particularly at the highest doses of P applied (Fig. 7). The analysis of percent VAM infection showed that VAM root colonization decreased with higher P doses, but a significant increase in infection was observed compared to the first harvest (Fig. 6). In ail three soils, plants mainly absorbed inorganic P corresponding to that extracted with 0.5 N NE&F (Fig. 8). The amount of P utilized from this fraction was higher in the Mayor and Curra soils, where the absorption increased with increased P addition. Inorganic P extracted with 0.1 N NaOH was also used, although to a lesser extent. No absorption of P by plants from the organic P-fractions was noted (Fig. 8), but the methodology followed may not detect changes in the organic P in a short-time period.
. __ 8
a
8
0
*
8
0
lO9%765432100 P-uptake(mglpotl
8
3
3
a
AWES
. 1
I
01
0.2
03
a4
0.5 % P
Fig. 4. Relationship between dry wt and %P and P-uptake of the shoots in Mayor (0). Anton (m) and Curra (A) soils.
P absorbed by mycorrhizal red clover plants
,
t 05 %
OL
t,
03
02
,
I
,
*
I
,
,
6.5
,
I
(
001236567090
01
P
P-uptake &t/pot)
Fig. 5. Relatjonship between %VAM
infection and %P and P-uptake of the shoots in Mayor (e)b Anton (8) and Curra (A) soils.
Table 3. Statisticalsignificance of red clovergrowthand P nutritionparametersin the three soilsas relatedto increasingP doses.First harvest Significance Soil Shoot dry WI (8 pot-‘1 Shoot P% Shoot P-uptake (mg pot-‘)
Phosphorus P
P < 0.01 P < 0.01
I.20 0.09
P co.01
P < 0.01
P < 0.01
2.99
increasing P addition enhanced plant growth at both harvests in all three soils studied. The highest P
dose did not result in a significantly increased yield respect to the P3 dose, thus confirming the predictions for P fertilizer needs by the McLean er al. method (1982).
r
LSD Interaction
P
DISCUSSfON
100
Interaction
(al
Differences in plant growth with increasing P supply were probably related to P-retention characteristics of the three soils, but a major role of the native VAM fungal populations in improving P-uptake by colonized roots, particularly at the lowest phosphate treatments, can not be discarded. VAM fungi have been shown to increase crop productivity and Puptake when colonizing roots grown in P-deficient soils or when low to moderate P doses are applied (Hayman, 1983). The mycorrhizal effect can not be established in our experiment, but the results on VAM infection and plant growth make us think that the symbiosis may have been effective at the lowest P doses or when no P was added, particularly in the Mayor soil, which reached the highest yields although showing the highest P retention properties (Fig. 2). Nevertheless, yields obtained with the highest P doses may have been a direct effect of P addition
20 400 600 800 1040 Phosphate appliedhg P/kg so11 I
0 Fig. 6. Effect of P addition on %VAM infection for Mayor (0). Anton (m) and Curt-a (A) soils. (a) 1st harvest, (b) 2nd harvest. 4 indicates that the increment in %VAM infection between the 1st and the 2nd harvest is significantly different at the 5% level.
200
LOO 600
800
1000
Phosphate applied(mgP/ Kg so11 I Fig. 7. Effect of P addition on root fresh wt for Mayor (a).
Anton (m) and Curra (A) soils. 0. n , A 1st harvest. 0, CJ, A 2nd harvest.
MARIA
66
J.
SAWZ
and JUSTO AR~XE.V
P-fertilizers are quickly adsorbed onto the most active surfaces of acid soils, bearing di- or trivalent LOO (or ions (Al, Fe, Ca) (Russell, 1973): this is confirmed in * our results were applied P was mainly retained in the 300 ,f ,. inorganic P-fractions extracted with NH,F (P bound F w :. to Al) and NaOH (P bound to Fe). These fractions are the principal sources of orthophosphate ions to the soil solution, as the plant and VAM fungi deplete available P. Our study of P absorption by plants agrees with this, since inorganic P extracted with NH,F was the fraction mainly utilized (Fig. 8). Vesicular-arbuscular mycorrhiza may have greatly contributed to the absorption of this fraction when the lowest P doses were applied, as Ross and Gilliam (1973) found that this fraction was better used by mycorrhizal than non-mycorrhizal plants. It is known g 200 that both fungal hyphae and roots take up phosphate from the labile pool of the soil (Mosse ef al., 1973), % 100 but Cress et nl. (1979) reported that fungal hyphae have a greater affinity than roots to phosphate. 0 Differences in P-affinity between VAM species may Pl P2 P3 Pl P2 P3 Pl P2 P3 Pl P2 P3 be related to their ability to take up the phosphate reIeased from NH, F- and NaOH-extracted inorganic Phosphate apptted P-fractions, particularly in soils with high PFig. 8. Inorganic P (P,) and organic P (P,,) utilized by plants retention. The fact that no utilization of organic P by from the soil fractions extracted with 0.5 N NH,F and 0. I N plants was detected further supports that only labile NaOH. (a) Mayor, (b) Anton and (c) Curra soils. 88 P P is readily available both to plant and VAM fungi, utilized. jr indicates that the utilization is significant at the at least in a short term. 5% level. The higher P-adsorption characteristics of the Mayor soil may have caused a greater dependence of red clover on mycorrhiza, and the presence of a rather than of mycorrhiza. Parfitt ef al. (1982) found that mycorrhiza had no effect at high levels of added highly effective VAM population may account for the P and Rangeley et al. (1982) observed that ~~~~~~~rn enhanced plant growth compared to the other two repens only responded to VAM infection when P soils. concentrations in the shoots were below [email protected]% Acknowledgemenrs-We thank Dr D. S. Hayman for This value was associated in our work .with VAM critical and grammatical revision of thr manuscript. colonization of IO-30% of the root, probably too low to account for the growth effect recorded in the first REFERENCES harvest. The decrease in VAM root infection on adding P Abbott L. K. and Robson A. D. (1982) The role of vesicular-arbuscular mycorrhizal fungi in agriculture and fertilizer has been widely reported in mycorrhizal the selection of fungi for inoculation. Ausfruliun Journal studies, this response being characterized by a maxiof Agticulrurat R&&h 33, 3891108. mum infection value when no P is applied and a Arines J. and Alvarez M. T. (19811 Contribucibn al estudio progressive decrease in VAM colonization as P applidel f&for0 en suelos icidos. i. Caracteristicas de adcation increases (Gianinazzi-Pearson, 1985). The ineorcibn y fracciones de P en una tierra parda de1 noroeste ternal P concentration of the plant, as a result of a de Esparia. Turrialha 31, 217-226. high P level in the soil, has been claimed as the factor Bloom P. R. ((981) Phosphorus adsorption by an aluminium-peat complex. Soil Science Sociery of’cimrrica controlling mycorrhizal colonization (Menge er al., Journal 45, 267-212. 1978). In this respect, at the first harvest, we found Bolan N. S. and Abbott L. K. (1983) Seasonal variation in inhibitory P concentration values in the plant for infectivity of vesicular-arbuscular mycorrhizal fungi in VAM infection to be 0.20425% and over, correrelation to plant response to applied phosphorus. Austrasponding to the addition of more than 2OOpg P g-’ lian Journuf of Sod Research 21. 208-210. to the soil. Therefore, this amount of soluble phosBray R. H. and*Kurtz 1. T. (1945) Determination of total phate marks the limit for P fertilization in the soils inorganic and available forms of phosphorus in soils. Soil studied if optimal benefits from P inputs and VAM Science 59, 39-45. infection are to be obtained. Chang S. C. and Jackson M. L. (1957) Fractionation of soil phosphorus. Soil Science 84, 133-11-1. P concentration of the plant was probably reCress W. A., Throneberry G. 0. and Lindsay D. L. (1979) sponsible for the increase in VAM colonization from Kinetics of phosphorus adsorption by mycorrhizal and the first to the second harvest at some P doses, nonmycorrhizal tomato roots. Plant Physiology 64, particularly in Mayor and Anton soils (Fig. 6). 484-487. despite the fact that root growth was higher (Fig. 7) Day P. R. (1965) Particle fractionation and particle-size and might have induced a dilution effect in the analysis. In Merhodr of Soil Analysis (C. A. Black et al., infection. According to Menge et al. (1978). P conEds), pp. 545-566. American Society of Agronomy, centration of the plant should have been lower to Madison. allow the higher percentages of infection recorded at Dick W. A. and Tabatabai M. A. (1977) An alkaline oxidation method for determination of total phosphorus the second harvest. NH&F-P.
\a3H-P,
NHLF-?a
NoOH-P.
P absorbed by mycorrhizal red clover plants in soils. Soil Science Society of America Journal 41, 51 l-514. Fox R. L. and Kamprath E. J. (1970) Phosphate sorption isotherms for evaluating the phosphate requirements of soils. Soil Science Society of America Proceedings 34, 902-907.
Gianinazzi-Pearson V. (1985) Mycorrhizal effectiveness in phosphate nutrition: how, when and where? In Proceedings of the 6th North American Conference on Mycorrhizae (R. Molina. Ed.), pp. 15&154). Bend, Oregon.
Giovannetti M. and Mosse B. (1980) An evaluation of techniques for measuring vesicular-arbuscular mycorrhizal infection in roots. New Phytologisr 84.489-500. Hayman D. S. (1983) The physiology of vesiculararbuscular endomycorrhizal symbiosis. Canadian Journal of Bofany 61, 944-963.
Hedley M. J., Stewart J. W. B. and Chauhan B. S. (1982) Changes in inorganic and organic soil phosphorus fractions induced by cultivation practices and by laboratory incubations. Soil Science Society of America Journal 46, 970-976.
Kamprath E. J. (1970) Exchangeable aluminum as criterion for liming leached mineral soils. Soil Science Society of America Proceedings
34, 252-254.
Lim L. L. and Cole A. L. J. (1984) Growth response of white clover to vesicular-arbuscular mycorrhizal infection with different levels of applied phosphorus. New Zealand Journal of Agricultural- Research 2;. 587-592.
McLean E. 0. (1965) Aluminium. In Merhoa!s of Soil Analysis (C. A.‘Black et al., Eds). pp. 978997. American Society of Agronomy, Madison. McLean E. 0.. Oloya T. 0. and Mostaghimi S. (1982) Improved corrective fertilizer recommendations based on a two step alternative usage of soil tests. I. Recovery of soil equilibrated phosphorus. Soil Science Sociery of America Journal 46, 1193-l 197.
Mehlich A. (1978) New extractant for soil test evaluation of phosphorus, potassium, magnesium, calcium, sodium, manganese and zinc. Soil Science and Plum Analysis 9, 477492.
Menge J. A., Steirle D., Bagyaraj D. J., Johnson E. L. V.
67
and Leonard R. T. (1978) Phosphorus concentrations in plants responsible for inhibition of mycorrhizal infections. New Phyrologist 80, 575-578. Mosse B.. Hayman D. S. and Arnold D. J. (1973) Plant growth responses to vesicular-arbuscular mycorrhiza. V. Phosphate uptake by three plant species from P-deficient soils labelled with ‘*P. New Phyrolonist 72. 809-815. Mugambi M. (1983) Soil organic mat& both a source and a sink for phosphorus in Kenya soils. Third fnremarional Congress on Phosphorus Compounds, October, pp. 587-59 I. IMPHOS, Brussels. Olsen S. R., Cole C. V.. Watanabe F. S. and Dean L. A. (1954) Estimation of available phosphorus in soils by extraction with sodium bicarbonate. U.S. Dept of Agriculture, Circular 939. ParIitt R. L., Hart P. B. S., Meyrick K. F. and Russell M. (1982) Response of ryegrass and white clover to phosphorus on an allophanic soil, Egmont black loam. New Zealand Journal of Agricultural
Research 25, 549-555.
Phillips J. M. and Hayman D. S. (1970) Improved procedures for clearing roots and staining parasitic and vesicular-arbuscular mycorrhizal fungi for rapid assessment of infection. Transactions of the Brirish Mycological Sociefy 55, I 59- I6 I. Rangeley A., Daft ht. J. and Newbould A. (1982) The inoculation of white clover with mycorrhizal fungi in unsterile hill soils New Phyfologist 92, 89-102. Ross 1. and Gilliam J. W. (1973) Effect of Endogone mycorrhiza on phosphorus uptake by soybeans from inorganic phosphates. Soil Science Society of America Proceedings
87, 237-239.
Russell E. W. (1973) The sources of plant nutrients in the soil: phosphate. In Soil Conditions and Plum Growrh, 10th ed. pp. 555603. Longman, London. Sharpley A. N. and Smith S. J. (1985) Fractionation of inorganic and organic phosphorus in virgin and cultivated soils. Soil Science Society of America Journal 49, 127-130.
Tiessen H., Stewart J. W. B. and Cole C. V. (1984) Pathways of transformations in soils of different pedogenesis. Soil Science Sociery of America Journal 48, 853-858.