Interactions Between P, AE and Glomus

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Pedobiologia 50 (2006) 413425

Interactions between phosphorus availability andan AM fungus (Glomus intraradices) and theireffects on soil microbial respiration, biomass andenzyme activities in a calcareous soil

Fayez Raiesi, Mahmoud Ghollarata

Soil Science Department, Faculty of Agriculture, Shahrekord University, P.O. Box 115, Shahrekord, Iran

Received 14 March 2006; received in revised form 10 August 2006; accepted 18 August 2006

KEYWORDS

Berseem clover;Calcareous soils;Interactive effects;Mycorrhiza;

Microbial activity;P fertilization

SummaryThe interactions between soil P availability and mycorrhizal fungi could potentiallyimpact the activity of soil microorganisms and enzymes involved in nutrient turnoverand cycling, and subsequent plant growth. However, much remains to be known ofthe possible interactions among phosphorus availability and mycorrhizal fungi in therhizosphere of berseem clover (Trifolium alexandrinum L.) grown in calcareous soilsdeficient in available P. The primary purpose of this study was to look at theinteraction between P availability and an arbuscular mycorrhizal (AM) fungus(Glomus intraradices) on the growth of berseem clover and on soil microbial activityassociated with plant growth. Berseem clover was grown in P unfertilized soil (P)and P fertilized soil (+P), inoculated (+M) and non-inoculated (M) with themycorrhizal fungus for 70 days under greenhouse conditions. We found an increasedbiomass production of shoot and root for AM fungus-inoculated berseem relative touninoculated berseem grown at low P levels. AM fungus inoculation led to animprovement of P and N uptake. Soil respiration (SR) responded positively to Paddition, but negatively to AM fungus inoculation, suggesting that P limitation maybe responsible for stimulating effects on microbial activity by P fertilization. Resultsshowed decreases in microbial respiration and biomass C in mycorrhizal treatments,

implying that reduced availability of C may account for the suppressive effects of AMfungus inoculation on microbial activity. However, both AM fungus inoculation and Pfertilization affected neither substrate-induced respiration (SIR) nor microbialmetabolic quotients (qCO2). So, both P and C availability may concurrently limit themicrobial activity in these calcareous P-fixing soils. On the contrary, the activities ofalkaline phosphatase (ALP) and acid phosphatase (ACP) enzymes respondednegatively to P addition, but positively to AM fungus inoculation, indicating that

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AM fungus may only contribute to plant P nutrition without a significant contributionfrom the total microbial activity in the rhizosphere. Therefore, the contrastingeffects of P and AM fungus on the soil microbial activity and biomass C and enzymesmay have a positive or negative feedback to C dynamics and decomposition, andsubsequently to nutrient cycling in these calcareous soils. In conclusion, soilmicrobial activity depended on the addition of P and/or the presence of AM fungus,which could affect either P or C availability.& 2006 Elsevier GmbH. All rights reserved.

Introduction

Soil microbial activity including soil respiration(SR) and enzyme activities, and the size ofmicrobial biomass, have been shown to depend onP fertilization and the presence of AM fungi in thesoilplant system (Amador and Jones, 1993; Wrightand Reddy, 2001; Wamberg et al., 2003; Lopez-

Gutierrez et al., 2004; Baligar et al., 2005;Marschner and Timonen, 2006). Phosphorus fertili-zation may affect soil microbial respiration andbiomass, especially soil enzymes, with variableresults depending on the soil P status. Phosphorusadditions resulted in increased microbial respira-tion in soils with low P contents, but not in soilswith high P contents (Amador and Jones, 1993;Smith, 2005). In contrast, P fertilization had aninhibitory effect on microbial respiration andsubstrate-induced respiration (SIR) in a pine forestfloor, while no effects on microbial metabolicquotients (qCO2) were detected (Thirukkumaran

and Parkinson, 2000). Increasing levels of soilapplied P significantly reduced acid phosphatase(ACP) activities and resulted in lower arylsulfataseand urease activities in acidic infertile upland soilsunder white clover cover (Baligar et al., 2005).Similarly, application of phosphate decreased ac-tivities of phosphatase, sulfatase, and urease(Haynes and Swift, 1988). Higher P concentrationsmay also depress the activity of some soil enzymesunder natural conditions. In a wetland soil, Ploading negatively influenced only the activity ofalkaline phosphatase (ALP) while other soil en-

zymes remained unaffected (Wright and Reddy,2001). The association of plants with mycorrhizalfungi can have strong influences on the responses ofmicrobial activities to P fertilization, due to thefact that these fungi are able to enhance Pavailability to and uptake by plants (Smith andRead, 1997). Following inoculation, AM fungi mayfurther influence microbial population and activity,and consequently nutrient dynamics in the soilthrough the release of organic compounds. Thereare many positive or negative interactions betweenAM fungus and soil microorganisms (see reviews by

Bonkowski et al., 2000; Hodge, 2000; Johanssonet al., 2004; Jones et al., 2004). Various studiesindicated that AM fungus may alter the populationcomposition (Bansal and Mukerji, 1994; Andradeet al., 1997; Vazquez et al., 2000) and activity ofsoil microorganism (Wamberg et al., 2003; Langleyet al., 2005), likely due to quantitative andqualitative changes in root exudation of colonized

plants occurring in the rhizosphere (Hodge, 2000;Barea et al., 2002). For example, root colonizationby AM fungi reduced the exudation of sugars, aminoacids and other organic compounds from the roots(Graham et al., 1981; Schwab et al., 1984; Bansaland Mukerji, 1994).

SR is one of the least studied microbial processesassociated with AM fungi. Yet, the influence ofmycorrhizal inoculation on SR is variable, anddepends on experimental conditions and themethodology involved. In the rhizosphere of peaplants (Pisum sativum) inoculated with Glomusintraradices, SR was negatively or positively

affected by fungi, depending on the growth stageof the plant (Wamberg et al., 2003). The negativeeffect was apparently due to the change in carbonflow from plant to fungal hyphae and thereforehigher anabolism occurred by fungus (Wamberget al., 2003). Mycorrhizal inoculation increasedmicrobial SR in sunflower rhizosphere before plantmaturity, due to the mycorrhizal stimulation ofplant growth (Langley et al., 2005). However, whenshoots were removed, mycorrihzal sunflower hadlower SR than the corresponding non-mycorrhizalplants. AM fungi may also influence other microbial

properties of the soil. Van Aarle et al. (2003)reported an increased microbial biomass andbacterial activity in the presence of AM fungalhyphae in a limestone soil. In contrast, Kim et al.(1998) showed that inoculation of tomato withGlomus etunicatum had no effects on total soilmicrobial biomass C (MBC). In the Entisols andVertisols under savannas, lower P mineralizationand higher microbial immobilization were asso-ciated with higher AM fungus colonization (Lopez-Gutierrez et al., 2004). Therefore, AM fungi maydirectly (Zhu and Miller, 2003) or indirectly (Langley

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F. Raiesi, M. Ghollarata414


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et al., 2005) contribute to soil C and N dynamics. Ithas been suggested that AM fungus could influenceC dynamics by transferring a large quantity of plantassimilates to fungal hyphae (Bago et al., 2000),but its significant role depends on other factorssuch as the kinds of hyphae produced and theresidence time of accumulated hyphal residues

(Zhu and Miller, 2003). On the other hand, AM fungiproduce glomalin (a glycoprotein), which could bea recalcitrant pool of soil C (Wright and Upadhyaya,1996, 1998; Rillig et al., 2001). It shows resistanceto microbial decomposition (Wright and Upad-hyaya, 1998; Rillig et al., 2001), and could lowersoil microbial activity. The activity of AM fungus inthe mycorrhizosphere could be a source of differ-ent soil enzymes required for biochemical reac-tions. There are various reports indicating that soilenzyme activities, such as phosphatases, dehydro-genase, urease, protease and beta-glucosidase are

increased by AM fungus inoculation (Kothari et al.,1990; Bolan, 1991; Kim et al., 1998; Vazquez et al.,2000; Caravaca et al., 2003). However, AM fungushad no effect on the activity of soil phosphatases(Van Aarle et al., 2003).

The dilemma is that while P fertilization gen-erally stimulates soil microbial activities, AMfungus inconsistently depress microbial populationand activities. Indeed, discrepancies may stemfrom differences in soil P availability and fromvariations in the extent of root colonization by theAM fungi, which would impose different carbondemands on the plants. However, relatively little is

known of the interactive effects of P fertilizationand AM fungus on microbial activity in calcareoussoils low in available P (Raiesi, 2006).

It is hypothesized that AM fungus may counteractthe stimulating effects of P fertilization on soilmicrobial processes including respiration, biomassand enzyme activities. The main objective of thisstudy was: (1) to study the interaction between Pfertilization and AM fungus on the growth ofberseem clover; and (2) to identify the importanceof P fertilization and AM fungus interactions on soilmicrobial biomass and enzymatic activity in the

rhizosphere of berseem clover.

Materials and methods

The interaction between P fertilization and theAM fungus (G. intraradices Schenk and Smith) onberseem clover (Trifolium alexandrinum L.) growth(including biomass production, N and P uptake) andits importance for some soil microbial properties(including respiration, biomass and enzymes) in the

highly P-fixing calcareous soils with low C contentsis not well established. Hence, a greenhouseexperiment was conducted to address the inter-active effects of P fertilization and AM fungi on

microbial activity in the rhizosphere of berseemclover. A 22 factorial experiment consisting oftwo levels of P fertilization and two treatments ofthe AM fungus arranged in a randomized completeblock design with eight replicates. The P fertiliza-tion treatments were P unfertilized soil (P) having10 ppm available P and P fertilized soil (+P) having30 ppm P achieved using KH2PO4 salt. The AM fungustreatments consisted of mycorrhizal plant (+M) andnon-mycorrhizal plant (M) as control. The soilselected for this study was taken from the surfacelayer (030 cm) of a clover field in the southern partof Shahrekord, the capital of Chaharmahal va

Bakhtiari, Iran. The soil is calcareous developed inlimestone with a clay loam texture and low inorganic C content (0.39%) and in plant available P(10ppm). A representative soil sample was air-dried, and passed through a 2-mm sieve forlaboratory analysis. Chemical parameters of the30-cm surface layer are reported in Table 1.

Experiment description

Plants were grown in plastic pots for 70 days in a

greenhouse at Agricultural Research Station inShahrekord. Before planting, seeds were surface-sterilized with sodium hypochlorite solution for5 min and rinsed with sterile distilled water. In agrowth chamber, seeds were placed on moist filterpapers in Petri dishes and germinated in the dark at25 1C. Five 3-day-old seedlings of uniform size(1.52 cm) were transferred into plastic pots (30-cm diameter 30cm depth) containing 4 kg ofautoclaved field soil-washed sand mixture (1.5:1ratio). After transplant, plants were inoculatedwith the AM fungus G. intraradices, in the form of a

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Table 1. Main characteristics of the soil used for thepot experiment

pH 8.03ECe 0.12 dS m1

CEC 24 cmol(+) kg1

Organic carbon 3.9 mgg1

Total N 0.28 mg g1

P (Olsens) 10 mg kg1

CaCO3 410mgg1

Sand 240 mg g1

Silt 460 mg g1

Clay 300 mg g1

AM fungi and P effects on soil microbiology 415


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mixture of spores, soil, external mycelium and rootfragments obtained from pot cultures. Twentygrams of inoculum (ca. 250 g per pot) were placedin each planting hole about 1 cm below the roots.The plants were inoculated with an effective strainof Rhizobium leguminosarum bv. trifolii 7 daysafter transplanting and irrigated with distilled

water. The average air temperature in the green-house was 2530 1C. Plants were grown undernatural light. At the end of experiment, plantswere harvested and separated into roots andshoots. Shoot materials were dried at 60 1C for 2days to determine dry weights. Roots were hand-washed to remove soil particles, then dried at 60 1Cfor 2 days and weighted. The ratio of shoot to rootwas calculated for each treatment. Roots werewashed and preserved in 50% ethanol for determi-nation of the extent of mycorrhizal root coloniza-tion. The percentages of root length colonized

(RLC) by mycorrhizal fungi (% colonization) andplant RLC was estimated by the grid-line intersectmethod (Giovanetti and Mosse, 1980) after clearingthe root systems with 10% (w/v) KOH at 70 1C for 4 hand staining with trypan blue (Phillips and Hayman,1970; Koske and Gemma, 1989). Shoot biomass wasanalyzed for the P and N concentrations using themethods described by Baruah and Barthakur (1997).Finally, soil samples were collected and stored at4 1C for the determination of soil microbiologicalproperties. All measurements were started within 2days of sample collection.

SR

SR was measured in a laboratory incubationexperiment for 38 days using the method describedby Anderson (1982) and Alef (1995). A moist sampleof 50g was placed in 0.750 l plastic jars, andmoistened by adding distilled water to a soilmoisture content corresponded to about 70% ofwater holding capacity. Five containers without soilwere considered as blanks. All jars were keptovernight in the dark at 25 1C prior to incubation. A

plastic vial containing 10 ml of 0.5 M NaOH, for CO2trap was placed in the jars, and replaced with afresh NaOH 2, 4, 6, 8, 11, 13, 15, 18, 21, 24, 28, 32,34 and 38 days after the start of the incubation.Upon replacing the NaOH solution, the jars wereopened and samples were re-aerated to supplyadequate oxygen. The evolved CO2 was trapped inNaOH and the excess alkali was titrated with 0.25MHCl after precipitating the carbonate with 15%BaCl2 solution. SR was calculated as the accumu-lative CO2-C evolved from the soil. All measure-ments were run in four replicates.

Microbial biomass C

Soil MBC was estimated using the chloroform-fumigation incubation method adapted from Jen-kinson and Powlson (1976). Two 20-g subsamplesfrom the respiration experiment were placed in 50-ml glass beakers. One beaker was fumigated with

ethanol-free chloroform in a vacuum desiccator for24 h at room temperature in the dark. The chloro-form vapor in the desiccator was removed by threerepeated evacuations. Both fumigated and unfumi-gated soils were re-inoculated with 0.1 g moist soil.All beakers were placed in 0.750 l plastic containerswith a vial containing 10 ml of 0.5 M NaOH for CO2absorption and a vial containing 10 ml of distilledwater to keep soil moisture constant. The contain-ers were closed tightly and incubated at 25 1C inthe dark for 10 days. The evolved CO2 wasdetermined as described above. The MBC was

calculated as

MBC CF CU

kc,

where MBC is the microbial biomass C, CF is theevolved CO2-C from fumigated soil, CU is theevolved CO2-C from unfumigated soil (the flush ofCO2) and kc is recovery factor equivalent to 0.45(Jenkinson and Ladd, 1981). All measurementswere carried out in four replicates.

Basal and substrate-induced respiration

For the measurement of the soil basal respiration(BR), a moist soil sample equivalent to 100 g (oven-dry basis) was placed in a 0.750 l plastic jar,adjusted to 60% of its water holding capacity andpre-incubated for 24 h in the dark at 25 1C. Theevolved CO2 was measured every 6 h for 5 days asdescribed above. The BR was expressed as mg CO2-C produced per kg soil per day. The metabolicquotient, qCO2, was calculated by dividing BR (theaverage CO2-C respired during 48 h, mg CO2-C h

1)by microbial biomass C, and expressed as mg CO2-C g1 Cmic per hour (Anderson and Domsch, 1990).

For measuring SIR, soil samples (100 g dry weightequivalent) were treated with 5 ml of 3% glucosesolution and then incubated at 251C, and theevolved CO2 was measured 6 h after glucoseaddition (Chen et al., 2002). The measurementswere carried out in four replicates.

Assay of soil enzymes

The activity of the ALP and ACP enzymes weremeasured by the methods described by Tabatabai

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(1994). A fresh soil sample of 1g was placed in50 ml test tube, to which one drop of toluene and4 ml of modified universal buffer (pH 11 for ALP andpH 6.5 for ACP) was added. The samples wereincubated with p-nitrophenyl phosphatase at 37 1Cfor 60min. After filtration, the yellow colorintensity was measured by spectrophotometer at

420nm. The enzyme activities were expressed asmg p-nitrophenol (PNP) released per gram soilwithin 1 h. The measurements were carried out inthree replicates and the activity of enzymes wasaveraged for all three replicates.

Statistical analysis

The effect of treatments was determined byfactorial two-way analysis of variance (SAS InstituteInc., 1999). Mean values were separated byprotected Fishers least significant difference

(LSD). Differences were considered significant onlywhen P values were lower than 0.05, unless statedotherwise. The relative change (%) of a variablewas calculated from (CT)/C l00, where T is themeasured value in the treated soils and C is themeasured value in the control (unfertilized oruninoculated) soil.

Results

Plant growth and nutrient uptake

There was no AM fungus colonization in roots ofuninoculated clover. The results of analysis ofvariance (Table 2) indicated that the treatmentsaffected most growth and nutritional parameters ofthe clover. Most plant variables were influenced bythe interactions between the two factors, Pfertilization and mycorrhizal inoculation. AM fun-gus colonization tended to be significantly lower(44%) in mycorrhizal plants grown at high P level(Table 3). Similarly, P additions decreased thelength of the colonized roots by 48% (Table 3). AM

fungus inoculation and P fertilization had a sig-nificant effect on shoot and root biomass (Table 2).Shoot and root dry weights were generally higherfor mycorrhizal than non-mycorrhizal plants, butshoot weight did not differ significantly betweenmycorrhizal and non-mycorrhizal plants grown athigh P levels (Table 3). Phosphorus addition led toenhanced root and shoot biomass production only inuninoculated treatments. The ratio of shoot to root(S/R) tended to increase with inoculating cloverwith AM fungus at low P level, while this effect wasnot observed at high P level (Table 3). The main

effects of AM fungus treatment and its interactionswith P level on N and P uptake were statisticallysignificant (Table 2). Shoot N uptake in treatmentswithout P and AM fungus was significantly lowerthan that in other treatments. In addition, the maineffect of AM fungus on N uptake was significant onlyin the treatments without P additions. Phosphorusfertilization and AM fungus inoculation resulted in asignificant N uptake by berseem clover. AM fungusinoculation increased shoot P uptake at both lowand high P levels, whereas the added P increasedshoot P uptake only in treatments without AMfungus.

Microbial activity and biomass

The cumulative microbial respiration from theincubated soils of mycorrhizal and non-mycorrhizalplants without P addition and fertilized with P areshown in Fig. 1. SR during the first 7 days of theincubation was similar for all treatments. However,a significant (Po0:05) difference in SR was ob-

served among the treatments afterwards (Fig. 1).Generally, SR decreased in mycorrhizal treatments,but it increased in P fertilization treatments.Results of analysis of variance showing the sig-nificance of the main treatment effects and theirinteractions on microbial soil properties are pre-sented in Table 4. AM fungus treatments signifi-cantly lowered the rate of soil respiration while Pfertilization increased SR rates (Po0.05, Fig. 2a).However, there was no interaction between thetwo factors on this parameter (Table 4). Neither AMfungus nor P fertilization affected SIR (Table 4).

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Table 2. Significance of the main treatment effectsand their interactions based on factorial ANOVA(F-values) with two factors (phosphorus and mycorrhizae)on berseem clover responses

Plant response Effect

Phosphorus

(P)

Mycorrhizae

(M)

PM

Colonization(Colon.)

11.72** 146.2*** 11.72**

Root lengthcolonized (RLC)

5.07* 49.1*** 5.06*

Shoot dry weight(shoot d.w.)

29.41*** 98.1*** 67.56***

Root dry weight(root d.w.)

36.51*** 94.0*** 33.88***

Shoot/root (S/R) 4.0ns 5.77* 12.49**

N uptake 3.86ns 21.01*** 4.73*

P uptake 3.34ns 110.1*** 15.01**

*

Po0:

05;**

Po0:

01;***

Po0:

001; ns, not significant



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Soil microbial biomass C (MBC) in non-mycorrhizaltreatments was significantly higher than that inmycorrhizal treatments at high P level (Fig. 2c). Incontrast, P fertilization had no significant effect onsoil microbial biomass C (Table 4). Results indicatedno main treatment effects of P fertilization and AMfungus, and their interaction on microbial meta-

bolic quotients (Table 4 and Fig. 3a). As a generaltrend, there was higher ALP activity in mycorrhizaltreatments regardless of soil P conditions. How-ever, P fertilization depressed the activity of ALP inboth non-mycorrhizal and mycorrhizal treatments.Surprisingly, the activity of this enzyme was notaffected by an interaction of P fertilization and AMfungus. The main treatment effects of P fertiliza-tion and AM fungus, and their interaction onthe activity of ACP was statistically significant(Table 4). AM fungus increased the activity of ACPonly at low P level, whereas P fertilization

decreased it regardless of AM fungal treatments(Table 4). Results also showed that AM fungus effecton ALP activity is more striking than on ACP activity(Fig. 3b).

Discussion

Phosphorus and AM fungus effects onberseem growth and nutrient uptake

Phosphorus addition clearly resulted in the

enhancement of plant growth and nutrient uptakein the absence of AM fungal inoculation in theseP-deficient soils. This is in agreement with previousresults obtained from most experiments (Azcon andEl-Atrash, 1997; Graham and Abbott, 2000). Incalcareous soils, a large proportion of P is found asprecipitated calcium-phosphate minerals, whichare insoluble and unavailable to plants in theshort-term (Strom et al., 2005). Consequently, Pfertilization may frequently lead to increased cropgrowth and production. Mycorrhizal colonizationand the improved P and N uptake increased growth

of both roots and shoots. Berseem clover inocu-lated with G. intraradices had higher shoot androot dry weights than non-mycorrhizal clover,particularly at low P levels. The beneficial effectsof AM fungus inoculation of most natural andagricultural plants grown under field and controlledconditions are widely recorded throughout theliterature (see Smith and Read, 1997; Rao andTak, 2001; Azcon et al., 2003; Ryan and Angus,2003; Martin and Stutz, 2004; Duponnois et al.,2005; Li et al., 2005). Most studies showed thatmycorrhizal plants grown under low P conditions

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Table

3.

EffectsofPfertilization(P

unfertilized;+P

fertilized

)andAMFinoculation(M

withoutinoculation;+M

withinoculation)

onrootcolonization;

shootandrootbiomassproductio

nandshootN

andPuptakebycloverafter70daysundergreenhousecon

ditions

Treatment

Colon(%)

RLC(m)

Shootd.w.

(gpot

1)

Rootd.w.

(gpot1)

Sh

oot/root

ratio

Shootnutrientuptake

N

(mgpot1)

P(mgpot1)

PM

0.0

0(0.0

0)

c

0.0

0(0.0

0)c

0.2

83(0.1

7)c

0.2

90(0.0

4)c

0.98(0.1

7)b

11.4

(1.0

5)b

26.0

(7.5

7)c

P+M

33.5

(4.2

0)

a

9.6

8(2.0

7)a

3.2

64(0.2

8)a

2.0

42(0.1

7)a

1.61(0.1

7)a

55.7

(1.4

9)a

698(79.9

)a

+PM

0.0

0(0.0

0)

c

0.0

0(0.0

0)c

2.5

30(0.3

1)b

1.6

29(0.1

2)b

1.57(0.1

1)a

35.8

(9.8

0)a

292(32.8

)b

+P+M

18.7

(0.9

4)

b

4.9

7(0.2

7)b

2.8

04(0.1

7)ab

2.0

67(0.0

9)a

1.37(0.0

5)a

54.5

(5.5

1)a

602(35.0

)a

LSD0.

05

6.6

4

3.2

2

0.5

07

0.3

48

0.27

20.3

144

Means7SE(n

8)incolumnsfollowe

dbythesameletterarenotsignificantly

differentat5%level.



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had higher biomass production compared to plantsgrown under high P conditions (Habte and Manju-nath, 1987; Graham and Abbott, 2000; Khaliq andSanders, 2000). In contrast, it has been reportedthat application of high amounts of P was requiredfor the beneficial effects of AM on wheat yield to beapparent in the highly P-fixing calcareous soils(Li et al., 2005). The higher shoot-to-root ratio inmycorrhizal plants could be due to the high

colonization and presumably as a result of produc-tion of large quantity of extraradical mycelium.Elevated shoot-to-root ratio is a common influenceof AM fungi on colonized plants (see Khalil et al.,1994, 1999; Smith and Read, 1997). This suggeststhat the AM colonized plants might allocate a largepart of their photosynthates to the mycorrhizalfungus. The higher shoot-to-root ratio (or lowerroot-to-shoot ratio) may also indicate the improvedplant nutrition with AM fungus association (Khalil

et al., 1999). Results indicate that mycorrhizalinoculation enhanced plant P and N uptake,particularly under low P conditions, which led toincreased shoot and root biomass. These datasuggest that berseem clover may have a relativelyhigh affinity (33.5%) to form a symbiotic associationwith G. intraradices. However, there is no pub-lished report about the extent of berseemsdependence of AM fungus in the literature.

Phosphorus effects on microbial biomass and

activity

The interactions between P fertilization andarbuscular mycorrhizal (AM) fungi on soil microbialactivity and biomass could be very important innatural and managed ecosystems. The main reasonis that the contrasting effects of P and AM on theoverall soil microbial activity and biomass may havea positive or negative feedback to nutrient cycling,which is essential for nutrient uptake and growth ofthe host plants in the long-term. Results of thisstudy indicated the highest SR under high P

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Table 4. Significance of the main treatment effectsand their interactions based on factorial ANOVA(F-values) with two factors (phosphorus and mycorrhizae)on soil microbial properties

Soil microbialvariable

Effect

Phosphorus(P)

Mycorrhizae(M)

PM

SR rate 4.52* 22.91** 0.02ns

Substrate-inducedrespiration (SIR)

1.37ns

0.01ns

0.4ns

Microbialbiomass C (MBC)

0.66ns 9.69** 0.04ns

Metabolicquotient (qCO2)

2.75ns 2.72ns 0.22ns

Acidphosphatase(ACP)

262.6*** 96.2*** 44.63***

Alkalinephosphatase(ALP)

62.2*** 6.72* 0.51ns

Po0:

05; Po0:

01; Po0:

001; nsnot significant.

Figure 1. Effects of P fertilization (P unfertilized; +P fertilized) and AM fungus inoculation (M withoutinoculation; +M with inoculation) on SR in the rhizosphere of clover after 70 days under greenhouse conditions. Each

point represents mean (n 8) and bars indicate SD.



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conditions, and the lowest SR in AM fungustreatments. This suggests that P and C availability

may concurrently limit the microbial respiration inthese calcareous P-fixing soils. Several studiesreported the stimulating effects of P fertilizationon soil microbial respiration (Amador and Jones,1993; Ilstedt and Singh, 2005; Smith, 2005), butThirukkumaran and Parkinson (2000) observed thatP additions had an inhibitory impact on SR, whichhas been ascribed to osmotic effects. There are twopossible mechanisms by which soil P status affectsmicrobial activity. The first mechanism is the directinfluence of P on soil microorganisms, as a nutrientrequired for microbial growth and maintenance,

which is always stimulatory. The second mechanismis the indirect effects through decreased rootexudations under high P conditions, which is ofteninhibitory. Root exudates are energy-rich com-pounds readily available to microorganisms, andcontribute to higher microbial activity in therhizosphere (Grayston et al., 1996; Kuzyakov,2002; Kuzyakov et al., 2003). Some studies showed

that plants grown under low P conditions increasedexudation of amino acids, reducing sugars andcarboxylic acids compared to plants grown underhigh P conditions (Ratnayake et al., 1978; Grahamet al., 1981; Schwab et al., 1983; Neumann andRomheld, 1999). Therefore, we could expect moreroot exudates under low P conditions, where weobserved decreased SR. However, our resultssuggest that without C limitation soil microbialrespiration is not affected by P availability, since Pfertilization had no effects on SIR. This furtherindicates that substrate availability is far more

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Figure 2. Effects of P fertilization (P unfertilized;+P fertilized) and AM fungus inoculation (M withoutinoculation; +M with inoculation) on (A) SR rate(mgCkg1 soild1), (B) substrate-induced respiration(SIR; mgCkg1 soil) and (C) microbial biomass C (MBC;mgCkg-1 soil) in the rhizosphere of clover after 70 daysunder greenhouse conditions. Means (n 8) 7SD fol-lowed by the same letter are not significantly differentbased on LSD test at 5% level.

Figure 3. Effects of P fertilization (P unfertilized;+P fertilized) and AM fungus inoculation (M withoutinoculation; +M with inoculation) on (A) the microbialmetabolic quotient (qCO2 (mgCkg

1 MBCd1) and (B) theactivity of acid (ACP) and alkaline (ALP) phosphatases (mg

p-nitrophenol g1 soilh1) in the rhizosphere of cloverafter 70 days under greenhouse conditions. Means (n 8)7SD followed by the same letter are not significantlydifferent based on LSD test at 5% level.



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important than P availability for microbial activity.The present results indicate lower, but insignif-icant, microbial biomass in P fertilized soils.Obviously, high variation in microbial biomasscontributed to lack of statistically significantdifferences. The small decline in microbial biomassC under high P conditions is probably due to the

lower input of plant derived and easily available C.However, the microbial metabolic quotient (qCO2)was not affected by P fertilization, suggestingmicrobial C efficiency would not depend on thesoil P status.

The results of our study indicate that P additionsubstantially decreased the activities of the ACPand ALP enzymes. It was indicated that theactivities of enzymes involved in P transformationare inversely related to P availability (Tadanoet al., 1993) and under P limited conditions itshigh demand, resulting in an increase in phospha-

tase activity, as occurred in low P conditions of thisstudy. It seems that any decrease in the availablephosphate may cause an overall increase inphosphatase activity. When plants are subjectedto P deficiency, secretion of ACP from roots is aregular reaction (Fox and Comerford, 1992; Gilbertet al., 1999; Richardson et al., 2001). However, it isunclear whether the release of ACPs into therhizosphere improves P acquisition.

AM fungus effects on microbial biomass andactivity

Mycorrhizas can affect the composition andactivity of soil microorganisms by changes in rootexudation patterns or fungal exudates (Marschneret al. 1997; Marschner and Timonen, 2006).Mycorrhizal colonization resulted in a significantdecline in SR. Similarly, the amount of soil micro-bial biomass C was lower in AM fungus treatmentscompared to treatments without AM fungus inocu-lation. However, we observed no effects of AMfungus inoculation on SIR and the microbial meta-bolic quotient (qCO2). The effect of AM fungus on

soil microbiological properties recorded in theliterature are inconsistent and complex, wherenegative (Christensen and Jakobsen, 1993; Wam-berg et al., 2003; Lopez-Gutierrez et al., 2004;Langley et al., 2005); positive (Van Aarle et al.,2003; Wamberg et al., 2003; Langley et al., 2005)or no (Kim et al., 1998) effects have beendetected, depending on other factors such as AMinoculum type, plants species, the plant growthstage, the kinds of hyphae produced, the residencetime of hyphal residues. AM fungus could influencemicrobial activity and biomass, most likely by

affecting root exudates quantitatively and qualita-tively in the hyphosphere. Changes in root exuda-tion and rhizodeposition following AM colonizationhave been reported, and include a reduction intotal sugars, reducing sugars and amino acids(Schwab et al., 1983, 1984; Bansal and Mukerji,1994). AM fungus may reduce the leakage of root

metabolites via decreased membrane permeability,especially under high P levels (Graham et al.,1981). We observed that SIR remained unaffectedby the presence of mycorrhizal fungi, furthersupporting that substrate availability in mycorrhi-zal treatments could be lower than in non-mycor-rhizal treatments.

It is also likely that the releases of glomalin byAM fungus could alternatively contribute to thedecreased microbial respiration and biomass C, asit appears to be relatively recalcitrant (Wright andUpadhyaya, 1998; Steinberg and Rillig, 2003). Since

glomalin was not measured, it is not valid to makeany conclusive comments on its effect on microbialactivity.

Mycorrhizal treatment clearly led to distinctiveincreases in the activities of enzymes involved in Pdynamics. Inoculation with AM fungus increased theactivity of ALP enzyme by 193% and 140% in low andhigh P levels, respectively. However, increases inthe activity ACP enzyme (143%) were significantonly at low soil P level. Our findings are inagreement with other studies indicating a higheractivity of various soil enzymes in the presence ofAM fungus (Kim et al., 1998; Rao and Tak, 2001;

Wang et al., 2006). There was a positive correlationbetween ACP activity and P uptake in low-P soils(Khalil et al., 1994), but Gianinazzi-Pearson et al.(1981) found no effect of mycorrhiza on ACPactivity. In contrast, Vazquez et al. (2000) reportedhigher enzyme activities in the rhizosphere ofmycorrhizal plants that may be due to the increasesin C and nutrient exudation from infected roots.However, the results of our study do not support it,since the microbial respiration did not increase inthe rhizosphere of mycorrhizal berseem. Phospha-tase activity in soil originates from many sources

including plant roots (Dodd et al., 1987; Dinkelakerand Marschner, 1992), mycorrhizal fungi (Tarafdarand Marschner, 1994; Joner et al., 2000) andbacteria (Tarafdar and Claassen, 1988; Ezawa andYoshida, 1994). Our data suggest that an AM fungussymbiosis contributed to the increase in theactivities of phosphatases. This may be a conse-quence of a direct contribution from the externalmycelium and an indirect effect of improved hostplant P status. Joner et al. (2000) proposed thathigher acid phosphate activity in mycorrhizaltreatments might be a result of a direct fungal

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leakage or an induced leakage by the plant roots.Additionally, decreased microbial activity in AMfungus treatments may suggest that soil micro-organisms do not contribute to the increase in theactivity of phosphatases. Therefore, AM fungus mayonly contribute to plant P nutrition without asignificant contribution from rhizospheric micro-

organisms.

Conclusions

Overall, we found several main treatment effectsof P fertilization and AM fungal inoculation andtheir interactions on berseem clover and soilmicrobial activities. An increased biomass produc-tion of shoot and root was evident, especially forAM fungus-inoculated berseem grown under low P

conditions. Phosphorus addition and AM fungusinoculation substantially improved P and N uptakeby the plant. It was also shown that P additionsstimulated soil microbial respiration, whereas AMfungus inoculation had a depressive impact on thismicrobial property. Increased microbial respirationand small decreases in biomass C in response to Pfertilization may indicate that P is a limiting factorfor microbial activity in the studied soilplantsystem. On the other hand, decreased microbialrespiration and biomass C in mycorrhizal treat-ments may imply that C availability could be alimiting factor for microbial activity and size. Our

data suggest that P and C availability may simulta-neously limit the microbial respiration in thesecalcareous soils, but it seems that C availability ismuch more important than P availability. Theactivity of ALP enzyme with AM fungus increasedby 193% and 140% in low and high P levels,respectively. However, increases in the activityACP enzyme (143%) were significant only at low soilP level. Our results suggest that while P fertiliza-tion generally stimulates soil microbial activities,AM fungus could depress microbial activities.Consequently, this contrasting effect may impose

a positive or negative feedback to soil C turnover,and thus to microbial transformations of essentialnutrients in these calcareous soils. The net re-sponse is yet unclear and needs further elucidation.We propose that a greater knowledge of interac-tions in the mycorrhizosphere and their effects onsoil microbial activity and subsequent nutrientavailability is even now desirable. This wouldenable the development of agricultural practicesto optimize plant growth and production in calcar-eous soils with low P, especially in naturallymycorrhizal plants fertilized with surplus P.

Acknowledgements

The authors gratefully acknowledge financialsupport from Shahrekord University. Thanks aredue to the two anonymous reviewers for providingvaluable and critical comments that significantlyimproved the manuscript.

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