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Research

Blackwell Science Ltd

Arbuscular mycorrhizas influence plant diversity and

community structure in a semiarid herbland

Patrick J. O’Connor, Sally E. Smith and F. Andrew Smith

Department of Soil and Water and The Centre for Plant Root Symbioses, Adelaide University, Waite Campus, PMB 1, Glen Osmond, South Australia 5064,

Australia

Summary

•

The contribution of arbuscular mycorrhizas (AM) to plant community structureand diversity is reported here in an annual herbland in southern Australia.

•

Mycorrhizal colonization was reduced in field plots by applying the fungicidebenomyl as a soil drench. The mycorrhiza-responsiveness of plant species wasassessed in intact soil cores containing the indigenous AM fungi and in a pot experi-ment using an isolate of

Glomus mosseae

.

•

Glasshouse experiments showed that

Medicago minima, Vittadinia gracilis

and

Velleia arguta

were highly mycorrhiza-responsive,

Salvia verbenaca

becamecolonized but exhibited no growth response to AM, and

Carrichtera annua

remaineduncolonized. There was no change in plant species richness in mycorrhiza-suppressed field plots but diversity increased owing to an increase in evenness.Treatment had no effect on community productivity and therefore there was no rela-tionship between mycorrhizal effects on diversity and productivity.

•

Mycorrhizal responsiveness was not a good predictor of species response tosuppression of AM in the field. The mycorrhiza-responsive species

V. gracilis

and

V. arguta

were not affected by reduced mycorrhizal colonization in fungicide-treatedplots, suggesting that competition from the mycorrhiza-responsive dominant

M. minima

offset the benefits of mycorrhizal association for these species.

Key words:

benomyl, community, diversity, evenness, functional group, arbuscularmycorrhizas.

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Author for correspondence:

Patrick J. O’ConnorTel: +61 (08) 8303 6514Fax: +61 (08) 8303 6511Email: patrick.oconnor@adelaide.edu.au

Received:

2 July 2001

Accepted:

12 November 2001

Introduction

Associations between plants and arbuscular mycorrhizal (AM)fungi are common in natural and agricultural ecosystems (Smith& Read, 1997). These associations contribute to plant nutrition,particularly plant uptake of poorly mobile soil nutrients suchas phosphorus (Smith & Read, 1997) and to improved waterrelations where water is growth limiting (Sanchez-Diaz &Honrubia, 1994). However, not all plant species form AMassociations, and not all AM plant species show clear nutritionalbenefits from colonization by mycorrhizal fungi under all growthconditions (Tester

et al.

, 1985; Fitter, 1986; Francis & Read,1995). Plant communities may often include species withvarying response to the presence of AM fungi. This variationmay range from nonmycorrhizal plants with benign orpossibly antagonistic interactions with the fungi (Francis &

Read, 1995), to highly responsive plant species with positivegrowth responses to AM association (Johnson

et al.

, 1997). Thecomposition of natural plant communities often includesplant species from across this response spectrum, and may bedetermined by differential contributions of the mycorrhizalfungi to individual plant species fitness.

In plant communities, the influence of mycorrhizas onindividual plant growth is further affected by interactionsbetween the individual plants. An established mycorrhizalmycelium is a potential resource and co-occurring plant speciesmay differ in their ability to compete for this resource despiteshowing individual responsiveness to mycorrhizal associationwhen grown separately in pots (Newman

et al.

, 1992). Theextent of host–plant benefit from mycorrhizas is also densitydependent (Koide & Li, 1991; Facelli

et al.

, 1999) and maybe influenced by neighbor competition (Hartnett

et al.

, 1993).

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Accordingly, changes in the abundance or size of individualsfrom one species may influence the abundance and size ofindividuals of the same or different species. The role ofmycorrhizas in regulating community structure will thereforedepend on the identity and functional characteristics of thespecies present, but can only be satisfactorily examined in intactcommunities.

Several studies have found that ecosystem processes such asprimary productivity are linked to species diversity (Naeem

et al.

, 1994; Tilman

et al.

, 1996, 1997; Hector, 1999). Whileincreasing species richness has been shown to increase primaryproductivity, species identity and functional group richnessare also important factors controlling productivity (Hooper& Vitousek, 1997; Tilman

et al.

, 1997; Symstad

et al.

, 1998;Wilsey & Potvin, 2000). Results from experiments on therelationship between plant species richness and productivityhave, however, been confounded by the ‘hidden treatment’effects discussed by Huston (1997) and Wardle (1999). Someresolution to the question of whether increased productivityis a function of species identity or diversity has been offeredby Wilsey & Potvin (2000). These authors suggest that diver-sity, measured as evenness (i.e. the distribution of abundanceor biomass among species in a community), can have a directeffect on plant productivity. However, these results come fromexperiments where evenness was manipulated in nonnaturalplots with very low species richness.

Grime

et al

. (1987) found that community structure couldbe significantly altered by mycorrhizal activity in species-richmixtures of plants in experimental microcosms. The presenceof mycorrhizas can increase floristic diversity (Grime

et al.

,1987) and species richness (Gange

et al.,

1990); however, thismay depend on the identity and mycorrhizal responsivenessof the dominant plant species (Bergelson & Crawley, 1988).Hartnett & Wilson (1999) have recently shown that suppres-sion of mycorrhizal fungi resulted in an increase in floristicdiversity in a tallgrass prairie, probably because the dominantC4 grasses in that system are more strongly responsive to myc-orrhizal colonization than the other species present.

Field-based studies of the involvement of mycorrhizasin structuring plant communities have been conducted inrelatively few plant ecosystems. Extensive tracts of openwoodland in semiarid Australia have been overgrazed, withthe understory reduced to annual herbland often dominatedby introduced species. The herb and grass species comprisingthese disturbed communities are thought to differ in theirresponse to mycorrhizal fungi. The influence of mycorrhizain controlling plant diversity may be vitally important insemiarid ecosystems, where communities can be subjectedto dramatic seasonal and interannual fluctuations, andmay rely on high biodiversity to maintain stability (Grime,1997).

This study was designed to test experimentally the relation-ship between mycorrhizal responsiveness of plants from asemiarid herbland and the influence of mycorrhizas on the

productivity and structure of this plant community. To testthis relationship, the mycorrhizal responsiveness of plant speciesfrom a semiarid herbland was assessed in a pot experiment andchanges in plant community structure in the field were meas-ured after formation of mycorrhizas had been suppressed byfungicide application.

Materials and Methods

Study area

This experiment was conducted at the Brookfield ConservationPark, a 5527 ha reserve situated at the southern-most extensionof the arid zone in South Australia (Lat. 34

°

19

′

18

′′

; Long.139

°

30

′

52

′′

). The site has been protected since 1971 when itwas purchased as a reserve for the Southern Hairy-nosed Wombat(

Lasiorhinus latifrons

). Long-term temperature data are avail-able from a nearby meteorological station at Waikerie, SouthAustralia. Temperatures range from an average maximum of32.8

°

C and minimum of 15.2

°

C in January, to lowestmonthly averages of 16.2

°

C maximum and 5.3

°

C minimumin July. The highest recorded temperature for the region is46.5

°

C. Rainfall is low and irregular, averaging 248 mm butvarying from 150 to 550 mm per annum. Rainfall in the yearof this study (1998) was 292 mm. This site has a droughtfrequency of 78%, calculated using the measure of Trumble(1948) and is characterized by extreme variation in theunderstory condition between years.

The study site is situated in the understory of low openwoodland and tall shrubland community dominated by sug-arwood (

Myoporum platycarpum

R. Br.), sheep bush (

Geijeralinearifolia

(DC.) J. Black) and bullock bush (

Alectryon oleae-folium

). The understory vegetation is characterized by annualherbs dominated by the introduced (‘weed’) species

Medicagominima

(L) Bartal. (Leguminosae; small erect herb with highpalatability),

Carrichtera annua

(L) DC. (Cruciferae; small erectherb with low palatability) and

Salvia verbenaca

L. (Labiatae;small herb with basal rosette and low palatability), and the nativespecies

Erodium crinitum

Carolin (Geraniaceae; small erect herbwith low palatability),

Velleia arguta

R. Br. (Goodeniaceae; smallerect herb, palatable) and

Vittadinia gracilis

(Hook. f.) N. Burb.(Compositae; small erect herb with low palatability). Thevegetation is heavily grazed by wombats and by kangaroos(

Macropus rufus

and

Macropus fuliginosus

) and regenerationof previously dense spear-grass (

Stipa nitida

Summerh. &C. E. Hubb.) is limited.

The soil is a clay loam (27% clay, 15% silt and 58% sand)to a depth of 20–25 cm, overlying thick calcrete over Miocenelimestone. The soil is generally low in nutrients with a pH of7.85, 0.85% organic carbon, 9.9 µg g

–1

available P (Colwell,1963), 8.6 µg g

–1

NO

3

-N and a cation exchange capacity (CEC)of 2.2 mEq 100 g

–1

. The dominant AM fungus species at thissite is

Glomus mosseae

(Nicol. & Gerd.) Gerdemann & Trappe(O’Connor, 2001).

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Field experiment design

Early in 1998, 21 1.5 m

2

plots were established in sevenblocks in open herbland (> 10 m to the nearest tree or shrub)by covering each with a wire cage (5 cm grid mesh, 1.5

×

1.0

×

0.5 m) to prevent grazing by large herbivores. Individuals ofthe short-lived perennial

Salvia verbenaca

were removed fromall plots by cutting at the base of the stem and swabbing withglyphosate herbicide (100 g l

–1

). This was done to leave nolive plants at the beginning of the growing season andgerminating

S. verbenaca

seedlings could then be treated asannuals in this experiment. Each plot was separated from theothers by a 1.5-m spacing and was divided into a central zoneof 1 m

2

and a surrounding buffer zone to reduce edge effectsfrom surrounding untreated vegetation. Plots in each block wererandomly assigned to one of the three treatments (mycorrhiza-suppressed, watered and unwatered control). Mycorrhiza-suppressed plots received the fungicide benomyl as a soil drench(9 g active ingredient (a.i.) in 15 l of water per plot). Wateredplots received 15 l of water per plot and control plots receivedno amendments. Treatments began after the first rains in Apriland were repeated every 2 wks until the end of August.

Benomyl was chosen for the mycorrhiza-suppressedtreatment as it has been shown to suppress colonization ofplant roots by glomalean fungi in field plots (Merryweather& Fitter, 1996). The use of benomyl is thought to be a con-servative approach to studying AM effects on plant com-munities (Wilson & Hartnett, 1997). The high rate offungicide used in this experiment was justified as previousexperiments had shown that at lower concentrations benomylis sorbed to particles in the surface layer of this soil (O’Connor,2001). Adequate suppression of the AM fungi requires perco-lation of the fungicide to greater than 5 cm below the soilsurface. It was necessary to include a control treatment receiv-ing only water, as well as an unwatered control, to determineeffects of additional water on the vegetation at this low rainfallsite.

Nontarget fungi

To test for any effects of benomyl application on nontargetsoil fungi, three soil samples (40 g) were collected on 8 Augustfrom the surface 10 cm of the buffer zone of each of the plots.Subsamples from each plot within a treatment were combinedto produce a composite sample for assessing the population offungi. Extraction of fungal propagules and dilution platingmethods followed a modified method of Alef (1995). Moistsoil (5 g) was mixed in 45 ml of 0.1% Na

2

H

2

P

2

O

7

for 1 h onan end-over-end shaker. One milliliter of this solution wasdiluted 1 : 10 in 0.1% Na

2

H

2

P

2

O

7

. Serial dilutions were madeusing this method to produce dilutions 10

–2

–10

–5

for solutionplating. Plates were made using half-strength potato dextroseagar (PDA) + 7.5 g agar l

–1

. Antibiotics were added beforepouring at rates of streptomycin 25 µg g

–1

and ampicillin

37.5 µg g

–1

. Three replicate plates of each soil dilution fromplots in each treatment were made by applying 100 µl of soildilutions 10

–2

–10

–5

to the sterile plates. Plates were incubatedat 25

°

C and observed after 1 wk and fungal colonies counted.Roots collected from field plots and intact cores for assessmentof mycorrhizal colonization were also examined for evidenceof pathogenic fungi.

Mycorrhizal colonization

Assessment of the effectiveness of benomyl for suppressionof AM fungi was made by collecting roots from field plotson 6 June and 8 August, 1998. On 8 August, five plantsfrom each of the most abundant species –

M. minima

,

C. annua

,

S. verbenaca

and

V. arguta

– were collected from the buffer zoneof each plot by excavating the whole root system (top 15 cmof soil). On 6 June, only roots of

M. minima

were collected asit was known to be colonized by AM fungi early in the growthseason (O’Connor, 2001). Roots from each species in eachplot were cut into approximately 1 cm lengths and clearedand stained in 0.05% trypan blue in lactic acid solution(Kormanick & McGraw, 1982). Root pieces were examinedfor mycorrhizal colonization at

×

40–100 magnification bythe grid intersect method (Giovannetti & Mosse, 1980) usinga light microscope.

Plant productivity and diversity

On 9 August 1998,

15 wks after treatments began (i.e. afterseven applications), a 20

×

20 cm subplot within each fieldplot was sampled for number and above-ground biomass of allplant species. The shoots were removed by cutting at the soilsurface and dried overnight at 80

°

C in an oven beforemeasurement of shoot dry weights. Relative biomass of eachspecies ((biomass per species

÷

total biomass of all species)

×

100) and relative number of each species ((number perspecies

÷

total number of all species)

×

100) were also calculatedfrom the number and biomass of each species in each plot.Plant number and biomass per species were also used tocalculate species richness (mean number of species per plot),diversity (Shannon

H

′

) and evenness (Shannon

J

).

Mycorrhizal responsiveness

Mycorrhizal responsiveness of the most common plants infield plots was assessed in two different ways. At the start ofthe experiment (20 April 1998), polyvinylchloride (PVC)cylinders 15 cm tall and 10 cm in diameter were driven intothe soil in an area adjacent to the experimental plot. Thecylinders were positioned in such a way that 10 cores werecollected for each of the major plant species (

V. gracilis

was notcollected); cores had a PVC cap on the bottom, which hadholes drilled in to allow free drainage. Seedlings were all at thecotyledon or first-leaf stage of growth at this early stage of the

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season. Cores were removed to a glasshouse and kept weededand watered for 12 wks. At the end of the first and third weeksin the glasshouse, half the cores for each plant species werelowered into a solution of 1.5 g l

–1

benomyl (a.i) for 5 min.These cores were designated as the mycorrhiza-suppressedtreatment. The remaining cores were lowered into waterfor an equal length of time and were designated as themycorrhizal treatment. Major plant species were also testedfor mycorrhizal responsiveness (

E. crinitum

was not tested asseeds were unavailable) by growing them in 400-g closed potsof Mallala soil–sand mix (Dickson

et al.

, 1999). Half of thepots for each species were designated as the mycorrhizaltreatment and contained 10% soil and root inoculum from

G. mosseae

(isolate NBR 4-1) cultured on

Trifolium sub-terraneum

cv. Mt Barker in the glasshouse.

Glomus mosseae

NBR 4-1 has been shown to produce a growth response in arange of plant species (P. J. O’Connor unpublished). Theother pots received an equal amount of nonmycorrhizalinoculum from pot cultures grown at the same time withoutmycorrhizal propagules. Pots were watered three times perweek and received 10 ml of Long Ashton solution (without P)once a week. Plants were harvested after 6 wks’ growth andthe extent of mycorrhizal colonization was assessed using themethods described above.

Statistical analysis

The unwatered control treatment was removed from all ana-lyses of the field experiment, as it showed no difference fromthe watered control treatment with respect to the relativeproportion of plant number and biomass per species. Wateredcontrol plots produced greater total above-ground biomassbut did not differ from unwatered controls for any measuresof community structure. All reference to controls hereafteris to the watered control treatment. Changes in communitystructure were analysed by

MANOVA

on plant number andspecies biomass, for the mycorrhiza-suppressed and controltreatments. Plots were used as replicates and fungicideapplication was used as the treatment. Plant species wereincluded as variates but only the six most abundant specieswere included, since all other species represented less than 2%each of the total plot biomass and were irregularly distributedbetween the replicates. Because the

MANOVA

indicated asignificant treatment effect for both plant number (Wilk’sLambda

F

= 4.17; df = 6,7;

P

= 0.051) and above-groundbiomass (Wilk’s Lambda

F

= 43.88; df = 6,7;

P

< 0.0001),protected ANOVA was performed on species-by-number andspecies-by-biomass to assess the influence of treatment onindividual species. Effects of suppression of mycorrhiza ontotal plot biomass, plant density, species richness, speciesdiversity and evenness were analysed using one-way

ANOVA

.All percentage colonization data were arcsine transformed

before analysis (Zar, 1999). Differences between treatmentswith respect to mycorrhizal colonization of plants from field

plots were tested by two-way

ANOVA

with species and treatmentas factors. Differences in colonization of plants in field-collectedcores and inoculated pots were tested using one-way

ANOVA

for each species. The number of colony-forming units of fungiisolated from field plots was analysed for differences betweentreatments by one-way

ANOVA

. Treatment differences wereseparated by Tukey’s HSD test for significantly differentmeans in all experiments.

Results

In the host plant responsiveness tests with field cores, averageroot length colonized was reduced to approximately 70% ofcontrols when cores were treated with benomyl (Table 1). Rootcolonization was significantly reduced (

P

< 0.05) in all thecases except

E. crinitum

(although colonization was reducedto 72% of controls for this species). The species shown inTable 1 accounted for more than 96% of total plot biomass inthe field experiment. Growth of the mycorrhizal host plants

M. minima

and

V. arguta

was significantly reduced in intactcores when colonization was suppressed. Growth of the hostspecies

S. verbenaca

and

E. crinitum

was not significantlyinfluenced by reduction in root colonization in benomyl-treatedcores, while the nonhost

C. annua

showed a significant (

P

< 0.01)growth increase when benomyl was applied (Table 1). In thehost plant responsiveness tests in inoculated pots (Table 1),there was no colonization of roots from any species inuninoculated pots. The growth of M. minima, V. arguta andV. gracilis was enhanced by inoculation with the mycorrhizalfungus G. mosseae (NBR 4-1). Salvia verbenaca showed nogrowth response to colonization by the mycorrhizal fungusand C. annua had a significant (P < 0.05) growth depressionin inoculated pots (Table 1).

The application of benomyl as a soil drench in field plotssuccessfully suppressed mycorrhizal colonization with respectto controls throughout the growth period. By early June,colonization of M. minima roots had been reduced in benomyl-treated plots to approximately 66% of the level in controls(P < 0.001; Table 2). Total colonization of the roots of themost abundant host plant species, M. minima, S. verbenacaand V. arguta just before the onset of flowering (8 August1998) was reduced in benomyl-treated plots to approximately50% of that in control plots (Table 2). Carrichtera annuaroots showed no signs of colonization by AM fungi.

In vitro tests on the population of culturable fungi in fieldsoils revealed that benomyl addition had a minor effect. Thenumber of colony-forming units of culturable fungi was notsignificantly different (P = 0.128) between control plots andbenomyl-treated plots (Table 3). Visual inspection of theroots of plants collected from field plots and intact coresrevealed no signs of pathogenic fungi or their effects.

Individual plant species response to suppression of AMfungi in field plots was only partly explained by mycorrhizalresponsiveness, as determined in intact cores or pots inoculated

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with G. mosseae NBR 4-1 (Table 1). The dominant species,M. minima, showed a significant (P < 0.001) reduction inabove-ground biomass in plots treated with benomyl relativeto control plots (Fig. 1a). Carrichtera annua and S. verbenacaboth showed significant (P < 0.001) increases in above-groundbiomass when benomyl was added to field plots (Fig. 1a).Only S. verbenaca showed a significant (P < 0.05) change in

plant density due to treatment. The number of S. verbenacaseedlings surviving was greater in benomyl-treated plots thanin controls (Fig. 1b). Total plant density also increased inbenomyl-treated plots (Table 4). None of the other speciespresent at the site showed any significant alteration (P > 0.05)in abundance or biomass due to benomyl treatment.

The structure of this semiarid herbland community wasaltered in benomyl-treated plots over the period of one grow-ing season. There was no significant difference in total plotbiomass between the benomyl-treated and control plots(P > 0.05; Table 4). Figure 2 shows the effects of mycorrhizalsuppression on the relative contribution of each species to theabundance and biomass of the field plots. Benomyl applica-tions resulted in a 62% reduction in the proportion of totalplot biomass made up by the mycorrhizal plant M. minima(Fig. 2a,b). Benomyl treatment also resulted in an increasein the proportion of plot biomass made up by the non-mycorrhizal plant C. annua (159%) and the facultatively

\

Root length colonized (%)

June 1998 August 1998

Medicago minima M. minima Salvia verbenaca Velleia arguta

Control 75.4 (3.1) 88.0 (2.0) 80.2 (5.6) 51.1 (7.5)Fungicide 49.5 (4.9) 46.7 (5.1) 45.2 (7.9) 24.7 (2.6)Percent reduction 65.6 53.1 56.4 48.3P-valuesTreatment < 0.001 < 0.001Species NA1 < 0.001Treatment × species NA ns2

1NA, not applicable; ns, not significant (P > 0.05).

Table 2 Per cent root length colonized with arbuscular mycorrhiza fungi in field plots treated with benomyl (fungicide) or water (control). Values represent the mean (SE) of seven replicates

Table 3 Number of colony-forming units (cfu) of fungi from field plots treated with benomyl (fungicide) or water (control). Fungi cultured in vitro by dilution plating. Values represent the mean (SE) of seven replicates

Plot treatment cfu g–1 soil

Control 8119 (2126)Fungicide 3750 (1549)ANOVA

P-value 0.128

Table 1 Growth response of single plant species to mycorrhizal colonization in intact cores and inoculated pots in a glasshouse. Each value represents the mean (SE) of five replicates

Field cores Plant species

Shoot dry matter (g plant–1) % Colonization

MGR1Control Fungicide Control Fungicide

Medicago minima 2.90 (0.24)a2 0.53 (0.23)b 80.8 (1.6)a 55.0 (3.0)b 448Carrichtera annua 1.32 (0.16)b 2.01 (0.05)a 0.0 (0.0)a 0.0 (0.0)a –34Salvia verbenaca 0.98 (0.07)a 1.04 (0.18)a 80.9 (3.2)a 57.1 (4.0)b –6Velleia arguta 0.89 (0.17)a 0.26 (0.15)b 75.1 (2.3)a 54.4 (8.6)b 245Erodium crinitum 1.20 (0.14)a 0.98 (0.25)a 61.3 (8.1)a 43.2 (5.1)a 22Vittadinia gracilis NA3 NA NA NA NA

Inoculated pots Mycorrhizal Control Mycorrhizal ControlM. minima 0.07 (0.01)a 0.04 (0.01)b 48.8 (12.3)a 0.0 (0.0)b 96C. annua 0.14 (0.02)b 0.18 (0.00)a 0.0 (0.0)a 0.0 (0.0)a –20S. verbenaca 0.10 (0.01)a 0.12 (0.03)a 79.5 (2.7)a 0.0 (0.0)b –13V. arguta 0.04 (0.01)a 0.01 (0.00)b 47.5 (7.0)a 0.0 (0.0)b 386E. crinitum NA NA NA NA NAV. gracilis 0.12 (0.02)a 0.01 (0.00)b 56.0 (4.3)a 0.0 (0.0)b 729

1MGR = Mycorrhizal growth response calculated as (mycorrhizal–non-mycorrhizal)/non-mycorrhizal × 100 (M – NM)/NM × 100. 2Different letters indicate significant difference (P < 0.05) between treatments for each plant species, as determined by Tukey’s honestly significant difference (HSD) test. 3NA, not available due to lack of seed or seedlings.

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mycorrhizal species S. verbenaca (1540%). There was no sig-nificant increase (P > 0.05) in the relative contribution of anyof the minor species (Fig. 2a,b).

The relative abundance of individual species was also alteredby application of benomyl. The relative abundance of M. minimawas reduced in benomyl-treated plots to 57% of controls(Fig. 2c,d). The relative abundance of C. annua remainedapproximately the same in different treatments, while S.verbenaca showed a 710% increase in relative abundance inbenomyl-treated plots with respect to controls (Fig. 2c,d).Differences in relative abundance for minor species were notsignificantly affected by treatment (P > 0.05).

Alterations in the abundance and biomass of individualplant species caused by suppression of AM fungi did nottranslate into a net change in species richness (Table 4).Plant species diversity and evenness increased in mycorrhiza-suppressed plots relative to controls (Table 4). Diversityincreased by approximately 29% and evenness by approx-imately 32% relative to controls. The minor species (allspecies excluding M. minima, C. annua and S. verbenaca)contributed equally (H ′ = 0.41) to the diversity of bothcontrol and fungicide-treated plots. Differences in diversitybetween the treatments came from changes in the contribu-tion of the three most abundant species. These species

0

20

40

60

80

100

120

140

M. minima C. annua S. verbenaca E. crinitum V. arguta V. gracilis

Abo

vegr

ound

bio

mas

s (g

m –

2)

(a)

*

*

*

0

500

1000

1500

2000

2500

3000

3500

4000

M. minima C. annua S. verbenaca E. crinitum V. arguta V. gracilis

Pla

nt d

ensi

ty (

m –

2)

(b)

*

Fig. 1 Response of major plant species (Medicago minima; Carrichtera annua; Salvia verbenaca; Erodium crinitum; Velleia arguta; Vittadinia gracilis) in field plots to suppression of arbuscular mycorrhiza (AM) fungi by benomyl application. Above-ground biomass (a) and plant density (b) response to benomyl (closed bars) compared with control plots (open bars). An asterisk above the bar indicates that the control is significantly different (P < 0.05) from the fungicide plots for that species (as determined by Tukey’s HSD test). Species shown accounted for > 90% of plot biomass; n = 7.

Table 4 Total above-ground dry matter production, plant density and plant species richness, diversity and evenness in field plots treated with benomyl (fungicide) or water (control). Values represent the mean of seven replicates

Community indicesAbove-ground dry matter(g m–2)

Plant density(m–2)

Plant species richness(species m–2)

Plant species diversity(Shannon H′)

Plant species evenness(Shannon J)

Control 157.3a* 6165a 8.29a 1.10a 0.53a

Fungicide 142.3a 8860b 8.29a 1.42b 0.70b

Different letters indicate a significant (P < 0.05) difference between control and fungicide-treated plots as determined by Tukey’s honestly significant difference (HSD) test.

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contributed 71% of H ′ in benomyl-treated plots but only63% in controls.

Discussion

Community structure was significantly altered by suppressionof mycorrhizal activity in this semiarid herbland. Plant speciesdiversity increased when mycorrhizal colonization was suppressedbecause growth of the highly mycorrhiza-responsive dominantM. minima was reduced and subordinate plant species werereleased from competition. The mycorrhizal responsiveness ofthe subordinate plant species in the community was not asimportant in determining species diversity as the mycorrhiza-responsiveness and subsequent strength of competition fromthe dominant plant species.

Treatment of field plots with benomyl successfully reducedmycorrhizal colonization without significantly altering popu-lations of other soil fungi. Benomyl treatments reduced totalAM colonization in intact cores to approximately 70% ofcontrols, and in field plots to approximately 50% of controls.Carey et al. (1992) showed that the effects of mycorrhizas werealtered even when mycorrhizal root colonization was reducedto an average of approximately 70% of that in untreatedcontrols. Smith et al. (1999) measured changes in individualplant growth, community structure and diversity whenmycorrhizal root colonization was reduced in benomyl-treatedplots to approximately 40% of that in untreated controls. The

method used in this experiment to detect change in the levelsof AM colonization was conservative and may not have detecteddynamic changes throughout the growing season. The effectof benomyl on AM formation can be dose dependant(Schreiner & Bethlenfalvay, 1997) and may depend on thetiming of application, soil heterogeneity and other site condi-tions (Pedersen & Sylvia, 1997). The use of root colonizationlevels as a surrogate measure of mycorrhizal activity may alsoresult in overestimation of the capacity of the AM associationto enhance P uptake. Larsen et al. (1996) showed that hyphalP-uptake was inhibited when benomyl was applied to anestablished hyphal network in soil.

Evidence linking reduction in the activity of AM fungi tochanges in the plant community is further supported by theabsence of symptoms of pathogenic fungi on the roots ofplants grown in field plots and intact cores. Pathogenic fungimay interact with AM fungi to influence plant growth andfecundity (Carey et al., 1992; Newsham et al., 1994; News-ham et al., 1995). However, the consistent response to AMfungi of individual plant species found in pot and intact coreexperiments indicates that any phytotoxic effects of benomylon plants, or fungitoxic effects on nontarget fungi, resulted innegligible effects on plant growth.

The decrease in mycorrhizal activity resulting from fungi-cide application caused significant changes in the abundanceand productivity of the dominant species and in relative con-tributions of a number of species to community composition.

Fig. 2 Species composition (%) of total above-ground biomass (a,b) and total number of individuals (c,d) in plots with arbuscular mycorrhiza (AM) fungi suppressed (benomyl) or AM fungi active (control). Species included are (clockwise from top) Medicago minima (dots); Carrichtera annua (vertical lines); Salvia verbenaca (black); Velleia arguta (dashes); Vittadinia gracilis (grey); Erodium crinitum (clear); and others (horizontal lines). Data represent the mean of seven replicates.

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The two most abundant species in control plots, M. minimaand C. annua, were affected differently by the reduction inmycorrhizal activity. The dominant, highly mycorrhiza-responsive plant M. minima, had reduced productivity, whilethe nonmycorrhizal C. annua increased in biomass inmycorrhiza-suppressed plots. These results are readily explainedas direct consequences of reduced mycorrhizal function inthe system. The strongly mycorrhiza-responsive M. minimashowed yield decline while the nonmycorrhizal C. annua wasreleased from any antagonism with the AM fungi. At the sametime, competition from M. minima was relieved. The increasein biomass and abundance of S. verbenaca is largely the resultof increased survivorship in mycorrhiza-suppressed plots.This is presumably due to reduced competition from thehighly mycorrhiza-responsive M. minima on the mycorrhizalbut poorly responsive S. verbenaca.

Negative growth response to interaction with AM fungi hasbeen previously observed for nonmycorrhizal plants (Francis& Read, 1995). The antagonism between C. annua and AMfungi was not large (> –34% mycorrhizal response) and isunlikely to account for all the increased biomass of this speciesin fungicide-treated plots. Sanders & Koide (1994) found asimilar relationship between the decline in biomass of themycorrhiza-responsive plant Abutilon theophrasti and the con-comitant increase in biomass of the nonmycorrhizal speciesAmaranthus retroflexus. The nonmycorrhizal species C. annua,and the poorly mycorrhiza-responsive species S. verbenacabehave in the same way in our study as the poorly mycorrhiza-responsive plant species in the study of Hartnett & Wilson(1999). The productivity of species with low mycorrhizaldependency in our semiarid herbland was not restricted byloss of mycorrhizal function after competitive release from thehighly mycorrhiza-responsive M. minima.

The change in biomass of the three most abundant speciesin our semiarid herbland accounted for all the increase indiversity (29%) in mycorrhiza-suppressed plots. However, equalcontributions of the minor species to diversity in mycorrhiza-suppressed or control plots indicates that loss of mycorrhizalactivity did not result in a significant decline in the highlymycorrhiza-responsive species V. gracilis and V. arguta. sinceneither of these minor species showed significant reduction inabove-ground biomass or number in response to mycorrhiza-suppression it is concluded that release from competition withthe highly mycorrhiza-responsive M. minima offset the cost oflosing mycorrhizal function. This indicates that while some ofthe minor plant species at this site were highly responsive tomycorrhiza, they may not have been as competitive asM. minima in acquiring resources from the external mycorrhizalmycelium. The high root density of the dominant plant,M. minima, in control plots may also have reduced mycorrhizalbenefit to subordinate species. The restricted growth of minorhost-plant species could have resulted from reduced mycorrhizalbenefit at high plant densities (Koide & Li, 1991; Koide,1991). Competition for nutrients in this plant community

may extend from competition between plants along themutualism–parasitism continuum (Johnson et al., 1997) tocompetition between plants with equivalent mycorrhizalresponsiveness but different capacities to exploit the symbiosisfor nutrient uptake or alleviation of water stress.

Reduction in the activity of mycorrhizal fungi resulted inan increase in plant species diversity owing to an increase inspecies evenness in this semiarid plant community. Thissupports the findings of Hartnett & Wilson (1999) that diver-sity (both richness and evenness) increased when mycorrhizalfungi were suppressed over several growing seasons in atallgrass prairie. Our results conflict with the microcosmexperiment of Grime et al. (1987) who showed that mycor-rhizal association increased plant species diversity (owing toincreased evenness), and the field experiment of Gange et al.(1990), who showed that reduction in mycorrhizal activity wascorrelated to a decrease in plant species richness. Mycorrhizalassociations were also shown to increase plant species diversityin macrocosms simulating North American old-field ecosystemsand increase evenness in microcosms simulating a Europeancalcareous grassland (Van der Heijden et al., 1998b). Ourresults support the hypothesis that mycorrhizal effects onplant species diversity are not absolute but depend on themycorrhiza-responsiveness of the component plant species,especially the dominant species (Bergelson & Crawley, 1988;Hartnett & Wilson, 1999).

Further support for this hypothesis comes from our study,where plant species richness was unchanged by suppressionof AM fungi, as was functional group richness. Decreasedmycorrhizal activity resulted in a redistribution of biomasswithin the community, i.e. an increase in evenness, but nochange in above-ground biomass of the community as awhole. This was also true in the studies of European calcare-ous grassland in microcosms (Van der Heijden et al., 1998b;but see Wardle, 1999), and tallgrass prairie in the field(Hartnett & Wilson, 1999). The fact that increasing diversityin our experiment did not coincide with increasing produc-tivity suggests that productivity may be linked to species orfunctional group richness (Naeem et al., 1994; Tilman et al.,1996; Tilman et al., 1997; Symstad et al., 1998; Hector,1999; Wilsey & Potvin, 2000), neither of which was changedin the current study. There is no evidence to suggest that theeffect of increasing diversity on plant productivity found inother studies (Tilman et al., 1997; Hector, 1999) can beexplained by increasing evenness (Wilsey & Potvin, 2000).Further study of the importance of functional group richnessin plant community productivity and stability, should con-sider mycorrhizal responsiveness as a key functional charac-teristic of plant species, particularly in disturbed and earlysuccessional communities.

The behavior of individual plant species was a strongdeterminant of community structure. Changes in speciescomposition following fungicide treatment of field plots alteredplant density. Changes in plant density were largely attributable

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to increased survivorship of seedlings of S. verbenaca inmycorrhiza-suppressed plots. This highlights the differentresponse of species to competitive release. There was anincrease in above-ground biomass of S. verbenaca related toincreased seedling survival, while C. annua showed increasedplot biomass without significant adjustment of survival. Thesedifferences may result in long-term effects on communitystructure not seen in one growing season, especially sinceS. verbenaca is a short-lived perennial.

The seasonal fluctuations in aridity and levels of stand-ing biomass may influence mycorrhizal activity in thissemiarid herbland. Severe grazing pressure may also affectbelow-ground allocation of carbon and hence interactionsbetween soil organisms and the plant community. Thepresent plant community reflects previous grazing distur-bance by large populations of kangaroos. Overgrazing hasfacilitated the establishment of several ruderal species includ-ing the nonmycorrhizal C. annua and the unresponsive hostplant S. verbenaca, probably as a result of the decline of nativeherbs and grasses, and the associated soil flora. While notinvestigated in this study, it has been observed that somespecificity between isolates of AM fungi and plant speciesfrom a grassland ecosystem (Van der Heijden et al., 1998a)exists. It is therefore possible that AM fungal communitiesenact a determinate role in plant community structure (Vander Heijden et al., 1998b). Restoration of the semiarid grass-lands of southern Australia will require ecological approachesto the re-establishment of some plant species, and greaterunderstanding of soil processes in disturbed and undisturbedsystems.

Acknowledgements

We wish to thank Anna Ziersch, Justin O’Connor, ShaezMortimer and Maria Roez for assistance in the field and EvelinaFacelli for advice and assistance with statistical analysis. Wealso thank reviewers of this paper for their useful suggestions.This research was supported by an Australian Post-graduateResearch Award to P. O’C.

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