plant community composition affects the biomass, activity and diversity of microorganisms in...

Plant community composition affects the biomass,activity and diversity of microorganisms in limestonegrassland soil

D. JOHNSONa, R. E. BOOTH

b, A. S. WHITELEYc , M. J. BAILEY

c, D. J. READa, J . P. GRIME

b & J. R. LEAKEa

aDepartment of Animal and Plant Science and bUnit of Comparative Plant Ecology, Department of Animal and Plant Science,

University of Sheffield, Sheffield S10 2TN, and cMolecular Microbial Ecology Section, CEH Oxford, Mansfield Road, Oxford OX1

3SR, UK

Summary

The diversity and functional type of plants can affect the microbial biomass in the soil, its respiratory

activity and the diversity of its bacterial population. We have studied these effects in microcosms of

reconstituted limestone grassland containing (i) a 12-species mixture of graminoids and forbs, (ii) a

monoculture of the sedge Carex flacca, (iii) a monoculture of the grass Festuca ovina, and (iv) similar soil

without plants. Microbial biomass was significantly greater in soil under monocultures of F. ovina than in

the other microcosms. Basal respiration was largest in the F. ovina and mixed-species treatments where

values were more than double those in the C. flacca and bare soil microcosms. The basal respiration was

strongly linearly related to plant productivity (r¼ 0.89). Analysis of the active bacterial population by

denaturing gradient gel electrophoresis of 16S rRNA revealed its diversity to be significantly greater in

the C. flacca and bare soil treatments than in the F. ovina or mixed-species microcosms. This suggests that

the functional type of plants has a strong influence on the composition of the bacterial community. We

hypothesize that the discriminating functional attribute leading to a reduction of bacterial diversity in

these microcosms was the presence in the F. ovina and mixed-plant communities of an active arbuscular–

mycorrhizal mycelium that is absent from bare soil and monocultures of C. flacca.

Introduction

Soil microorganisms have key roles in litter decomposition and

nutrient cycling in ecosystems. In addition, their biomass can

represent a significant component of the total labile carbon

(C), nitrogen (N) and phosphorus (P) in the soil. The activity

and biomass of microbial communities can be influenced by

several abiotic and biotic factors. Since virtually all soil

microbes ultimately depend on autotrophs for their supply of

C, the species composition and activity of the plant community

is likely to determine critically both the biomass and the

diversity of microbial species. Access to C derived from plants

may be either direct, as in the case of mycorrhizal fungi, or

indirect through the deaths of roots and shoots, root exud-

ation, sloughing of root cells and exudation and death of

mycorrhizal mycelia. Collectively these activities give rise to

local zones of C enrichment in the immediate vicinity of the

root surface, referred to as the rhizosphere, which favour

microbial proliferation in particular (Curl & Truelove, 1986).

Much effort has been devoted to characterization of the

microbial populations in the rhizospheres of individual plant

species, particularly in agricultural monocultures, and evi-

dence is emerging that a given plant supports a distinctive

bacterial community (see, e.g., Grayston et al., 1998). How-

ever, many such studies have used techniques that depend on

culturability of the organisms concerned and so are essentially

selective. They target only a small fraction of the total popula-

tion, often selecting preferentially for fast-growing Gram-

negative species (e.g. members of the g-proteobacteria; Smalla

et al., 1998). The potential to overcome these limitations has

been provided recently by the development of procedures such

as phospholipid fatty acid profiling and amplification of 16S

rDNA and rRNA, which enable characterization of the whole

microbial population (for a review see Head et al., 1998). These

methods pave the way, in turn, for an evaluation of the

relationships between diversity and function in populations

of soil microorganisms.

Correspondence: D. Johnson, School of Biological Sciences,

University of Aberdeen, Cruickshank Building, St Machar Drive,

Aberdeen AB24 3UU, UK. E-mail: [email protected]

Received 21 February 2002; revised version accepted 28 May 2002

European Journal of Soil Science, December 2003, 54, 671–677 doi: 10.1046/j.1365-2389.2003.00562.x

# 2003 Blackwell Publishing Ltd 671

As a result of the past emphasis on agricultural systems we

remain largely ignorant of the relationships between plant and

microbial diversity in semi-natural plant communities. How-

ever, because there are large qualitative and quantitative dif-

ferences both in the root exudates from individual plant

species and in the nutrient status, e.g. C:N ratios of their tissue

residues (Wardle et al., 1997), we can suppose that in perman-

ent grassland, in which plants are typically long-lived, the

individual species will sustain distinctive microbial popula-

tions. As a consequence, diverse plant communities are

hypothesized to sustain greater microbial diversity than mono-

cultures and these in turn more diversity than bare soil. We

have tested these hypotheses using outdoor microcosms

containing soil that had supported synthesized communities

comprising individual species, mixtures of species and no

plants for 4 years.

Materials and methods

Establishment of grassland microcosms

In 1997, soil was removed from the Ah horizon (up to a

maximum depth of 15 cm) of a species-rich undisturbed lime-

stone grassland in close proximity to the Buxton Climate

Change Impacts Laboratory (Buxton, Derbyshire, UK;

National Grid Reference SK 055 706). The soil is a Humic

Rendzina containing little carbonate (< 0.08%). The soil was

coarsely sieved (10mm) to remove stones and packed into free-

draining 60 cm� 60 cm� 15 cm deep acrylic boxes at the

University of Sheffield botanical gardens. Into each box,

except bare soil controls, 192 mature plants were transplanted

from the field in a 14� 14 grid (each corner remained empty).

Communities were assembled from plants representative of

three broad functional types common to limestone grasslands

(sedges, grasses and forbs) to give four levels of diversity up to

a maximum of 12 species (Carex caryophyllea Latour., C.

flacca L., C. pulicaris L., C. panicea L., Briza media L., Festuca

ovina L., Helictotrichon pratense (L.) Pilger, Koeleria

macrantha (Ledeb.) Schultes, Campanula rotundifolia L.,

Leontodon hispidus L., Succisa pratensisMoench, Viola riviniana

Rchb.). In the 12-species mixtures, planting was randomized

within the grids and between each replicate microcosm. Use of

mature transplants avoided the vagaries of seedling establish-

ment, enabled specific combinations of species to be estab-

lished, and led to the rapid development of a closed turf. It

also increased the likelihood that the established microbial

populations in the rhizosphere associated with the plants

were retained and as similar as possible to those in the natural

environment. Each treatment had four replicates assigned at

random within the experimental set-up and was maintained by

regular weeding. The experiments described in this paper used

four of the treatments, namely, bare soil, C. flacca only,

F. ovina only and a mixture of 12 species. We estimated above-

ground biomass in autumn 2000 and 2001 by cutting three

6-cm diameter circles from the microcosm boxes to soil level

and oven-drying and weighing them. Samples of fresh roots

were collected from each of the microcosms that supported

plants, and we determined the extent of root colonization by

arbuscular–mycorrhizal (AM) fungi using the line intersect

method (McGonigle et al., 1990) after staining with cotton

blue in lactophenol. The biomass and length of the roots

(Tennant, 1975) were measured in 10-mm diameter cores

from each of the microcosms.

Total N and C in the soil and �13C isotopic signatures were

determined on finely ground oven-dried bulk samples by

isotope ratio mass spectrometry (ANCA-GSL preparation

module connected to a 20-20 stable isotope ratio mass spectro-

meter; PDZ Europa, Middlewich, Cheshire). We determined

concentrations of P in soil using the molybdenum blue method

(John, 1970) after digesting 100mg dry soil in 1ml salicylic–

sulphuric acid. Soil pH was measured in distilled water with a

digital probe (2:1 water:soil ratio).

Measurement of basal respiration rates and microbial

biomass C

We measured respiration rates of soil microbial communities

using a sealed system ‘Respicond’ (Nordgren Innovations AB,

Umea) automated conductometric respirometer (Nordgren,

1988). Intact bulk soil samples were incubated at 22�C, and

respiration rates were logged every 30minutes for 8 hours.

Microbial biomass C (Cmic) was determined in sieved (2mm)

soil samples by substrate induced respiration (Anderson &

Domsch, 1978). We incubated a 10-g soil sample at 22�C

after adding glucose (using a previously optimized concentra-

tion of 10mg g�1 added in 2ml water), and respiration rates

were measured every 20minutes. The maximum rate measured

within the initial 4 hours of incubation was used to calculate

Cmic (Anderson & Domsch, 1978). We calculated the meta-

bolic quotient (qCO2) by dividing basal respiration by Cmic

(Anderson & Domsch, 1993).

Assessment of bacterial functional diversity by DGGE of

reverse transcribed 16S rRNA

Triplicate soil samples were removed from three microcosms

of each treatment (i.e. bare soil, Carex flacca, Festuca ovina

and a mixture of 12 species), bulked, sieved (2mm), and frozen

(�20�C). We analysed the bacterial community by denaturing

gradient gel electrophoresis (DGGE) following reverse tran-

scription polymerase chain reaction (RT-PCR) of extracted

16S rRNA. The procedure followed that of Griffiths et al.

(2000). Briefly, total nucleic acid was extracted from 1 g

fresh soil subsamples in Bio-101 Multimix 2 matrix tubes in

combination with a FastPrep FP120 bead-beating system with

0.5ml hexadecyltrimethylammonium bromide (CTAB) and

0.5ml phenol:chloroform:isoamyl alcohol (25:24:1; pH8).

After removing the phenol, we precipitated total nucleic acids

672 D. Johnson et al.

# 2003 Blackwell Publishing Ltd, European Journal of Soil Science, 54, 671–677

with a solution of 30 g polyethylene glycol 6000 in 100ml and

1.6M NaCl for 2 hours, followed by centrifuging (18 000 g) at

4�C for 10minutes. Pelleted nucleic acids were washed in 70%

ethanol and air-dried prior to resuspension in 50�l of RNase-

free Tris–EDTA buffer (at pH7.4).

Extraction of RNA was confirmed by agarose gel electro-

phoresis and staining with 1% ethidium bromide. We analysed

the variable helix 3 (V3) region of the amplified 16S rRNA by

DGGE as documented in Griffiths et al. (2000). The primers

targeted members of the Domain Bacteria to provide micro-

bial community diversity band profiles. Amplified 16S

fragments (approximately 200 bp) were analysed electro-

phoretically with a 10% acrylamide denaturant gradient gel

consisting of a 30–60% formamide gradient on a Dcode

(BioRad UK) apparatus. The DGGE profiles were digitized

after they had been stained with 1�gml�1 SYBRGold (Mol-

ecular Probes, OR). We determined the number of bands

present on each profile with Phoretix 1-D gel analysis software

(Phoretix, Newcastle-upon-Tyne).

Statistical analyses

Analysis of variance and Tukey multiple comparison tests

were used to determine statistical differences between means

of each microcosm composition. Soil C, N and C:N ratio and

basal respiration data were transformed to common loga-

rithms to equalize the variance of the distributions. Means

were back-transformed to obtain 95% confidence limits.

Results

Soil pH ranged from 6.2 to 6.5 and was significantly greater in

the 12-species mixture than in the bare soil microcosm (Table

1). Total soil C contents ranged from 0.51mg g�1 soil in the

bare soil microcosm to 0.72mg g�1 soil in the microcosm

containing 12 plant species (Table 1). The largest values,

0.68, 0.67 and 0.72mg g�1 soil, were in the C. flacca, F. ovina

and 12-species microcosms, respectively, and these were

significantly (P< 0.001) greater than the value from bare soil.

A similar pattern was seen in concentrations of soil N, which

ranged from 5.3 to 6.8mg g�1. The mean concentrations of soil

N in the C. flacca, F. ovina and 12-species microcosms were

6.0, 6.4 and 6.8mg g�1, respectively, and these were signifi-

cantly (P< 0.001) greater than the concentration of soil N in

the bare soil microcosms. The N concentration in the soil in

the microcosms containing 12 species was significantly greater

than in the microcosm containing a monoculture of C. flacca.

The C:N ratios ranged from 9.6 to 10.6, the smallest being

recorded in the bare soil and the greatest in the C. flacca

monoculture, although all the plant communities had signifi-

cantly larger values than in bare soil. The pattern of �13C

values of bulk soil was similar to that found for total C and

ranged from �26.3 to �27‰. Soil from the bare soil micro-

cosms was significantly more enriched in 13C, by between 0.52

and 0.69‰, than all the vegetated microcosms. Total concen-

trations of soil P ranged from 0.71 to 0.76mg g�1 and were not

significantly affected by plant community composition or by

the absence of plants (Table 1).

Roots from the F. ovina and 12-species mixture microcosms

supported significantly more AM colonization compared with

those from the C. flacca microcosms (Figure 1). Mean root

length colonized by AM fungi was approximately 70% in the

F. ovina microcosms, 58% in the 12-species microcosms and

4% in the C. flacca microcosms.

Microbial biomass C ranged from approximately 30 to

100�g g�1, most being measured in the F. ovina monoculture

(Figure 2a). In the 12-species mixture, C. flacca and bare soil,

Cmic was significantly less than in the F. ovina monoculture.

Basal respiration in C. flacca monocultures was 15mg CO2 g�1

soil and was twice as large as in bare soil (8mg g�1). In both

the F. ovina monocultures and 12-species mixture (Figure 2b)

it was twice as large again (32mg g�1). The qCO2 ranged from

75mg CO2-C g�1 Cmic hour�1 with bare soil to 150mg CO2-C

g�1 Cmic hour�1 in the 12-species mixtures (Figure 2c), but

because of the large variance between replicates none of

these differences was significant. The basal respiration rate of

soil was positively correlated (r¼ 0.89, P < 0.001) with above-

ground plant biomass (Figure 3).

Table 1 Soil pH, total carbon and nitrogen, C:N ratio, isotopic signature and total phosphorus of soil from the different microcosms (� 95%

confidence limits). Values sharing a letter are not significantly different (P> 0.05)

Composition of microcosm pH (H2O)

Total C

/mg g�1

Total N

/mg g�1 C:N ratio d13C /‰

Total P

/mg g�1

Bare soil 6.2b (þ0.1) 0.51b (þ0.52) 5.33c (þ0.15) 9.6b (þ9.9) �26.34b (þ0.04) 0.73a (þ0.06)

(�0.1) (�0.50) (�0.15) (�9.3) (�0.04) (�0.06)

Carex flacca 6.3ab (þ0.3) 0.68a (þ0.76) 6.03bc (þ0.16) 11.3a (þ12.2) �26.86a (þ0.33) 0.71a (þ0.04)

(�0.3) (�0.61) (�0.16) (�10.4) (�0.33) (�0.04)

Festuca ovina 6.3ab (þ0.3) 0.67a (þ0.73) 6.35ab (þ0.17) 10.5a (þ11.1) �26.88a (þ0.29) 0.76a (þ0.12)

(�0.3) (�0.61) (�0.16) (�10.0) (�0.29) (�0.12)

12 species 6.5a (þ0.2) 0.72a (þ0.88) 6.78a (þ0.18) 10.6a (þ11.3) �27.03a (þ0.27) 0.71a (þ0.05)

(�0.2) (�0.59) (�0.16) (�10.0) (�0.27) (�0.05)

Plant community composition and microbial diversity 673


Bacterial community diversity was determined by image

analysis of 16S rRNA band profiles. Significantly more

bands were identified on the DGGE profiles (P< 0.05) from

the bare soil and C. flaccamonocultures than from the F. ovina

and 12-species communities (Figure 4).

Discussion and conclusions

Soil microbial biomass and activity in the limestone grassland

soil was strongly influenced by the assemblages of plant species

in the microcosms. Our observations accord with those made

in several other studies that have demonstrated links between

both the productivity (Zak et al., 1994) and composition

(Groffman et al., 1996; Spehn et al., 2000) of a plant commu-

nity and the biomass of associated soil microbes. At the eco-

system level, Zak et al. (1994) found a positive linear

relationship between microbial biomass and above-ground

net primary production along a continent-wide gradient in

North America. In plots manipulated to have different plant

species richness, Spehn et al. (2000) showed that Cmic declined

as the number of plant species diminished.

In our study Cmic was 95% greater in the F. ovina than in the C.

flaccamicrocosms, and 230% greater in the grass than in the bare

soil system. The small value in the bare soil microcosms strength-

ens the view that microbial biomass and activity are determined

by the availability of recent (< 4years) inputs of C from plants.

Some measure of the potential for supply of this C in our micro-

cosms can be obtained from both the total plant productivities

(Figure 3) and the root biomass (data not shown). These were

greatest in the microcosms containing a monoculture of F. ovina

and 12 species, which supported both the largest microbial bio-

mass and the greatest basal respiration rates. Basal respiration

from the microcosms is a consequence of both microbial activity

and root respiration, for which the latter can contribute a sig-

nificant component (e.g. Boone et al., 1998). The relative contribu-

tion of roots to basal respiration is likely to depend not only

on their density but also on the plant species composition, the

amount of mycorrhizal colonization, and soil conditions.

Rhizodeposition of C can vary considerably between plant

species and may also reflect their habitat niche (Tyler & Strom,

1995). In particular, dauciform cluster roots characteristic of

many Carex species (Davies et al., 1973) are thought to be sites

of active organic acid production, as in the case of the mor-

phologically similar proteoid roots, which are known to have

profound impacts on microbial populations in the rhizosphere

(Wenzel et al., 1994).

80

90

b

Microcosm composition

a

a70

60

50

40

30

20

10

0C. flacca F. ovina 12 species

Per

cen

t roo

t len

gth

colo

nize

d

Figure 1 Arbuscular–mycorrhizal colonization of roots removed from

microcosms containing monocultures of Carex flacca or Festuca ovina

or a mixture of 12 species (� 95% confidence limits). Bars sharing a

letter are not significantly different (P> 0.05).

160

120

80

40

0

40

30

20

10

0

a

c

b

a

b

(a)

(b)

(c)

b

a

b

a

a a

a300

Met

abol

ic q

uotie

nt/m

g C

O2-

C g

–1 C

mic

hou

r–1B

asal

res

pira

tion

rate

/mg

g–1 h

our–1

Mic

robi

al b

iom

ass

carb

on (

Cm

ic)

/µg

g–1

250

200

150

100

50

0Bare soil C. flacca F. ovina 12 species


Figure 2 (a) Microbial biomass C, (b) basal respiration, and (c)

metabolic quotient in microcosms (� 95% confidence limits). Bars

sharing a letter are not significantly different (P> 0.05).



Links have also been sought between plant species composi-

tion and specific functional groups within microbial commu-

nities. Stephan et al. (2000), using Biolog microtitre plates,

showed that catabolic activity and diversity of the culturable

soil bacteria increased linearly with the logarithm of the number

of plant species. However, since these methods include only a

small fraction of the total microbial population (Smalla et al.,

1998; McCaig et al., 2001), it is difficult to interpret their rele-

vance to the community as a whole. Basal respiration rates and

Cmic that we measured were as great as or greater in the grass

monoculture than in the 12-species mixture, indicating that for

the entire microbial population the effects of plant species diver-

sity, as distinct from total plant biomass, may be small or absent.

Our use of 16S rRNA for the determination of microbial

diversity adds to our assessment of the effects of plant species.

In addition to providing a view of the whole rather than the

culturable components of the bacterial population, use of

RNA rather than DNA selectively detects the active compon-

ent of the microflora (Nogales et al., 2001). The image

analysis of DGGE bands of amplified 16S rRNA indicates

that active bacterial diversity was not related to plant diversity,

at least in the assemblages we used. However, the data suggest

that functional type of the plants, interpreted in the broad

sense of grass, sedge or a mixture of grass, forb and sedge,

had a much stronger effect on active bacterial populations

than did plant diversity. The greatest number of bands was

found in the bare soil and C. flacca, while the least occurred in

the F. ovina and 12-species mixture. Although the number of

bands does not relate directly to the number of species, it is

likely to be strongly correlated with the dominant community

members.

It is necessary to consider the possible basis for the observed

differences between the more active bacterial diversity in the

bare soil and C. flacca microcosms and reduced richness in the

F. ovina and mixed-species microcosms. In relation to their

known microbial associations, these two categories are most

readily distinguishable by their propensity for the formation of

mycorrhiza. Whereas neither the C. flacca nor the bare soil

system supported an active mycorrhizal mycelial system, both

the F. ovina and a large proportion of the 12 species in the

mixture were heavily colonized by AM fungi. In nature, soils

are normally intensively exploited by AM fungal mycelia and

these can yield several metres of mycelium per gram dry soil

(Miller et al., 1995). The nature of the relationship between

AM mycelial systems and soil bacterial populations remains

unclear, but there is some evidence for adverse effects on

00

30

Bas

al r

espi

ratio

n ra

te /µ

g C

O2

hour

–1 c

m–3

25

20

15

10

5

20 40 60 80

Shoot biomass /g m–2

100 120 140 160 180

Figure 3 Correlation between basal respiration

and shoot biomass in microcosms containing

bare soil (.), Carex flacca only (&), Festuca

ovina only (~), and a mixture of 12 species (^)

(r¼ 0.898).

20

aba

bb

0


Bare soil C. flacca F. ovina 12 species

4

8

12

Num

ber

of r

RN

A b

ands

16

Figure 4 Number of 16S rRNA bands from image analysis of DGGE

gels in microcosms (� 95% confidence limits). Bars sharing a letter are

not significantly different (P> 0.05).



bacterial numbers. Ravnskov et al. (1999) detected antagonis-

tic effects of the mycelia of Glomus intraradices upon selected

strains of pseudomonads, while Waschkies et al. (1994) showed

that inoculation of vines with AM fungi led to reductions in

the sizes of the general bacterial populations, particularly

Pseudomonas fluorescens. Other studies (e.g. Olsson et al.,

1998) have shown few qualitative impacts of AM fungi on

the composition of the bacterial population.

Although at this stage we cannot unequivocally ascribe the

differences between the two categories of microcosm to mycor-

rhizal effects, the presence of an active AM mycelium provides

a major and distinct route for C to flow from plants to the soil

microbial populations. Johnson et al. (2002) have recently

shown, using in situ 13CO2 pulse-labelling of permanent grass-

land, that more than 6% of the net C fixed by the turf is

respired within 24 hours by external AM mycelia, and this

does not include C incorporated into the mycorrhizal mycelial

systems. The allocation of significant amounts of recent photo-

synthate from plants to mycorrhizal mycelium, and the con-

tribution of that mycelium to fluxes of nutrients and C in the

soil, are likely to have a significant impact on soil microbial

populations. Again, it is possible that dominance of the AM

fungal system with its direct access to photosynthate may be

sufficient to eliminate some less specialized microbial groups.

The extent to which the observed reduction of microbial diver-

sity is attributable to the effects of the presence of AM fungi or

to the availability of substrates, as distinct from antagonism,

remains a question requiring further study.

Although our study has provided some insight into the effects

of plant diversity on microbial communities, there remain serious

gaps in our understanding of the linkages between plant and

microbial diversity. In particular, it is difficult to distinguish

between effects of plant composition and those of biodiversity,

especially when, as suggested here, the functional types of plant

have strong effects on soil microbial communities. There is

clearly a need to understand more fully the complexities of the

relationships between plant diversity and functional group,

mycorrhizal fungi and free-living microbial populations.

Acknowledgements

We thank Dr Warwick Dunn for analysis of the �13C signa-

tures and Irene Johnson for skilled technical assistance. This

work was supported by grants to J.R. Leake and M.J. Bailey

from the Natural Environment Research Council Soil Biodi-

versity Thematic Programme (grant numbers GST/02/2117

and GST/02/2136).

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