Growth, nutrition, and soil respiration of a mycorrhiza-defective
tomato mutant and its mycorrhizal wild-type progenitor
Timothy R. CavagnaroA,E, Adam J. LangleyB, Louise E. JacksonC, Sean M. SmuklerC
and George W. KochD
ASchool of Biological Sciences and Australian Centre for Biodiversity, Monash University, Clayton,
Vic. 3800, Australia.
BSmithsonian Environmental Research Centre, Edgewater, MD 21037, USA.
CDepartment of Land, Air and Water Resources, University of California Davis, One Shields Avenue,
Davis, CA 95616-8627, USA.
DNational Institute for Climatic Change Research, Box 5640, Northern Arizona University, Flagstaff,
AZ 86011, USA.
ECorresponding author. Email: tim.cavagnaro@sci.monash.edu.au
Abstract. The effects of colonisation of roots by arbuscular mycorrhizal fungi (AMF) on soil respiration, plant growth,
nutrition, and soil microbial communities were assessed using a mycorrhiza-defective tomato (Solanum lycopersicum L.)
mutant and its mycorrhizal wild-type progenitor. Plants were grown in rhizocosms in an automated respiration monitoring
system over the course of the experiment (79 days). Soil respiration was similar in the two tomato genotypes, and between P
treatments with plants. Mycorrhizal colonisation increased P and Zn content and decreased root biomass, but did not affect
aboveground plant biomass. Soil microbial biomass C and soil microbial communities based on phospholipid fatty acid
(PLFA) analysis were similar across all treatments, suggesting that the two genotypes differed little in their effect on soil
activity. Although approximately similar amounts of Cmay have been expended belowground in both genotypes, they may
have differed in the relative C allocation to root construction v. respiration. Further, net soil respiration did not differ between
the two tomato genotypes, but root dry weight was lower in mycorrhizal roots, and respiration of mycorrhizal roots per unit
dry weight was higher than nonmycorrhizal roots. This indicates that the AM contribution to soil respiration may indeed be
significant, and nutrient uptake per unit C expenditure belowground in this experiment appeared to be higher in mycorrhizal
plants.
Additional keywords: mycorrhizamutant, mycorrhizas, PLFA, respiration, roots, root respiration, Solanum lycopersicum.
Introduction
Soils play an important but poorly understood role in the global
carbon budget. Plant-derived C enters the soil as plant litter and
root exudates (Coleman et al. 1976; Butler et al. 2003), and is
released as CO2 by the respiration of roots and soil
microorganisms. As much as 50% of soil CO2 efflux is root-
derived (Rochette and Angers 1999; Kuzyakov and Domanski
2002), with contributions coming directly from root respiration
and indirectly from heterotrophic respiration of C originating
as root exudates, exfoliates (sloughed cells and root hairs), and
root debris (Ingham et al. 1985; Cheng 1996; Luo et al. 1996;
Kuzyakov and Domanski 2002; Langley et al. 2005). Roots may
also alter soil respiration via inorganic carbon fixation in the
root zone (Cramer et al. 1993).
The arbuscular mycorrhizal fungi (AMF) that colonise the
roots of most terrestrial plant species can influence soil carbon
processes in a variety of ways and at a range of scales from the
individual plant to the ecosystem (Rillig 2004). Primary among
these influences is improved plant nutrition (Marschner and Dell
1994; Smith and Read 1997) which can lead to increased
growth and soil C inputs. AMF can also stabilise soil
aggregates (Rillig 2004), and this may slow soil C turnover
and increase long-term C storage. AMF may also influence
rates of soil CO2 efflux from soils, both directly via their own
metabolism (Pang and Paul 1980; Peng et al. 1993; Valentine
and Kleinert 2007) and indirectly via their effects on root growth
and metabolism as well as by heterotrophic consumption of
AMF-derived C (Langley et al. 2005), for example, by
collembolans and nematodes (Gange 2000; Bakhtiar et al. 2001).
Colonisation of roots by AMF can alter plant C allocation
(Jakobsen and Rosendahl 1990) (depending on developmental
stage Mortimer et al. 2005), and stimulate plant photosynthetic
rates (Smith and Read 1997). Allocation of C to AMF may vary
with stage of plant development (Mortimer et al. 2005).Although
some studies have shown that colonisation of roots by AMF
can increase belowground respiration (Pang and Paul 1980; Peng
et al. 1993), others have suggested otherwise (Silsbury et al.
1983). Reasons for such differences remain unknown, although
CSIRO PUBLISHING
www.publish.csiro.au/journals/fpb Functional Plant Biology, 2008, 35, 228--235

 CSIRO 2008 10.1071/FP07281 1445-4408/08/030228
size differences betweenmycorrhizal and non-mycorrhizal plants
are likely an important factor in determining total belowground
respiration (Langley et al. 2005). Methodological artefacts may
also contribute to the observed variation in the effects of AMF
on belowground respiration. Sterilisation or fumigation of soils is
commonly used to establish controls in mycorrhizal studies, yet
can cause elimination of non-target members of the soil biota
(see Cavagnaro et al. 2006; for discussion). This may lead to
underestimation of the heterotrophic component of soil
respiration, and hence, overestimation of the mycorrhizal
contribution to soil respiration, with an uncertain influence on
total belowground respiration. To avoid such potential problems,
a mycorrhiza-defective tomatomutant (rmc) and its mycorrhrizal
wild-type progenitor (76RMYC+) (Barker et al. 1998) have been
used in both glasshouse and field settings to study the effects of
AMF on plant growth and nutrition (Gao 2002; Poulsen et al.
2005; Cavagnaro et al. 2006), plant ecology (Cavagnaro et al.
2004), and responses to elevated atmosphericCO2concentrations
(Cavagnaro et al. 2007b). The growth of the two genotypes has
been found to be very similar under a range of circumstances
(Cavagnaro et al. 2004, 2006; Poulsen et al. 2005), including
non-mycorrhizal conditions, suggesting that the mutation
affecting colonisation of rmc by AMF has no pleiotropic
effects on other plant processes (Cavagnaro et al. 2004).
Recently, Langley et al. (2005) used an automated respiration
monitoring system which measures soil respiration with a high
degree of temporal resolution, with and without plants and
AMF, over the entire course of a plant?s growth cycle. This
unique experimental system allows for averaging of diurnal
effects on soil respiration, and assessing the impacts of both
abiotic and biotic factors on soil respiration. Using this system,
we compared plant growth and nutrition, soil microbial biomass
and community profiles, and belowground respiration in
mesocosms, of rmc and 76R MYC+ tomato genotypes to
avoid disturbance of the wider soil biota (Cavagnaro et al.
2006). In order to influence the potential nutritional benefits
of mycorrhizal status, we crossed two levels of phosphorus
availability with the two tomato genotypes. Specifically we
addressed the following questions:
(1) does respiration of mycorrhizal and non-mycorrhizal root
systems of otherwise similar plants differ; and
(2) does the presenceof roots, colonisedbyAMFornot, alter soil
microbial biomass C (MBC) or microbial community
composition (PLFA profiles)?
Materials and methods
Soil collection, mixing and nutrient addition
The sandy loam soil used in this study was from the Epikom
series (loamy, mixed, superactive, mesic Lithic Haplocambids)
collected from a desert grassland 40 km north of Flagstaff,
Arizona, and similar to that used in earlier work (Langley
et al. 2005). The soil was chosen to facilitate root removal due
to its sandy texture, and because its low C content was expected
to decrease background heterotrophic respiration. The soil was
mixed in a 1 : 2 ratio with sand. Physiochemical properties of the
final soil : sand mix, including soil particle size distribution, pH,
exchangeable Na, exchangeable K, exchangeable Mg,
exchangeable Ca, total N, total C and DPTA extractable Zn,
Mn, Cu and Fe were determined (Table 1), by the DANR
Analytical Laboratory, University of California Davis
(http://danranlab.ucanr.org, verified 17 March 2008). K2HPO4
was mixed with the soil to establish high (7.5mg P added/g soil)
and low (1.5mg P added/g soil) P treatments. One hundred
millilitres of the appropriate P solution was added in a drop-
wise manner to 90 kg of soil, spread out on a plastic sheet.
The solution was then ?rubbed? into the soil by hand, and the
soil thoroughly mixed for several minutes. Final plant available
(Olsen) P concentrations in ?low P? and ?high P? treatments were
104
 2 and 113
 2mg P/g soil (mean
 s.e.), respectively, as
determined by the same laboratory. Although initial soil P
concentrations were higher than anticipated, the difference
between the two P addition treatments were significant, as
were plant responses to them (see Results). This soil type, and
other related desert soils, can be high in total P, although
available P is typically lower than found here (Kra?mer and
Green 1999). The soil was packed into plastic free-draining
pots. Each pot contained 4.5 kg of soil : sand mix, on a dry
weight basis. Eighteen pots of each P treatment were established.
Plants
Seeds of the mycorrhiza-defective tomato mutant (rmc), and
its mycorrhizal wild-type progenitor S. lycopersicum cv. 76R
(76R MYC+) (Barker et al. 1998), were surface-sterilised and
pre-germinated following Cavagnaro et al. (2006). Seeds
germinated within ~5 days, after which they were planted in
peatmoss in seedling trays and grown in a glasshouse for 26 days,
followedbyhardening in a lath house for 21days.These seedlings
were then used in the main experiment.
Experimental design
An experiment was established as a randomised complete
block design on 6 July 2004. There were two soil P
amendment treatments (high or low P, see above) and three
plant treatments: 76R MYC+, rmc, or plant free soil controls
(soil blanks hereafter), applied in combination, giving a total of
36 pots filled with soil. An additional six replicates were
Table 1. Soil characteristics of the soil : sand mix
Soil physical and chemical properties Mean 
 s.e. (n= 4)
Sand (%) 80.9 (0.25)
Silt (%) 10.4 (0.45)
Clay (%) 8.8 (0.5)
pH 6.7 (0.0)
Total N (%) 0.1 (0.01)
Total C (%) 0.3 (0.05)
C :N ratio 5.7 (0.2)
Exch. K (meq/100 g) 0.6 (0.02)
Exch. Na (meq/100 g) 0.1 (0.01)
Exch. Ca (meq/100 g) 9.5 (0.32)
Exch. Mg (meq/100 g) 2.6 (0.12)
Extractable Zn DPTA (ppm) 0.3 (0.03)
Extractable Mn DPTA (ppm) 21.3 (3.9)
Extractable Cu DPTA (ppm) 1.0 (0.06)
Extractable Fe DPTA (ppm) 40.9 (7.7)
P (Olsen), plant available, low P treatment (ppm) 104.2 (2.2)
P (Olsen), plant available, high P treatment (ppm) 113.4 (2.1)
The mycorrhizal contribution to soil respiration Functional Plant Biology 229
included in the experiment which consisted of pots without
plants or soil (chamber blanks hereafter). Following planting,
pots were placed in sealed belowground rhizocosms to monitor
soil respiration.
Automated respiration monitoring system
Soil respiration was monitored over the course of the
experiment using the automated respiration monitoring system
at Northern Arizona University. A detailed description of the
glasshouse layout and respiration chambers (rhizocosms) is
given by Langley et al. (2005). Briefly, immediately following
planting the pots were placed in rhizocosms, constructed of
air-tight, white PVC cylinders (50 cm height 13 cm
diameter). The top of each chamber was sealed around the
plant stem with a split rubber stopper with a centre hole and
gas-impermeable putty (Quibitac, Qubit Systems, Kingston, ON,
USA). Six pots from each of the six experimental treatments
(see above) and six completely empty pots (chamber blanks)were
eachassigned to1of42monitored rhizocosms.Aprogramwritten
and run in LabView (National Instruments, Austin, TX, USA)
controlled a Hewlett-Packard data acquisition system (HP3495)
that managed air handling and signal acquisition. CO2
concentration of air drawn from above the glasshouse and
delivered to the inlet port on each rhizocosm (reference [CO2])
wasmeasured using an infrared gas analyser (IRGA,Model 6262,
Li-Cor, Lincoln, NE, USA). The concentration of CO2 exiting
each microcosm was measured by the IRGA ~every 2 h (sample
[CO2]). A mass flow meter (Model 820, Sierra Instruments,
Monterey, CA, USA) measured the flow rate through each
container (~400 mL min--1). CO2 flux (soil respiration) from
rhizocosms was calculated following Eqn 1:
soil respiration ? flow rate
 ?sample ?CO
2
  reference ?CO
2
?:
?1?
Pots containing either soil or soil and plants were weighed
and watered regularly to maintain the weight corresponding to
11% gravimetric moisture. Once a week, plants were also
supplied with 30mL of a modified Long Ashton solution
minus P (Cavagnaro et al. 2001).
Plant sampling
Plants were destructively harvested 79 days after planting on
22September2004.The soil chambersweredisassembledand the
experimental pots removed. Plant shoots were immediately cut
at the soil surface and fresh weights determined. The shoots
were separated into stems and leaves, dried at 60C, weighed,
and ground for nutrient analysis. Shoot and fruit B, Ca, Fe, K,
Mg,Mn,Na, P andZn contentswere determined on plantmaterial
that was microwave-digested with nitric acid (Sah and Miller
1992) and analysed by ICP-AES (Thermo Jarrell Ash Corp.,
Franklin,MA,USA). Stable isotope ratios, and concentrations, of
C andNweremeasured bymass spectrometry at theUniversity of
California Davis Stable Isotope Facility (http://stableisotopefaci
lity.ucdavis.edu/, verified 17 March 2008) as described by
Cavagnaro et al. (2006).
Soil was removed by cutting the pot away from the soil mass,
which retained its form. Soils from 0 to 8 cm and 8--30 cm
depth were mixed separately by hand for 30 s, subsamples
were taken, and soil analyses performed as outlined below.
Roots were extracted from each zone using a combination of
dry picking with forceps and wet sieving, and root length
determined using a root length scanner (Comair, Melbourne,
Victoria, Australia) and the grid line intersect method (Tennant
1975). A subsample of roots was dried at 60C and dry weights
determined. Mycorrhizal colonisation of roots was determined
using a modified grid-line intersect technique as described
previously (Cavagnaro et al. 2006).
Soil sampling
Soil from the 0--8 cm soil zone was subsampled as follows.
Samples for PLFA community analysis were taken first;
aggregates (5--10mm in diameter) were collected and
transferred to tubes for storage at --20C. Phospholipid fatty
acid (PLFA) analysis was performed using a modified
chloroform-methanol extraction (Bligh and Dyer 1959; Bossio
and Scow 1998) with transesterification of the polar lipid
fraction containing the phospholipids (Guckert et al. 1986), as
described by Cavagnaro et al. (2006). Only the low P samples
were used for PLFA analysis because these treatments were
expected to show the greatest mycorrhizal dependence.
Duplicate subsamples from each soil sample (25 g moist soil)
were analysed for microbial biomass C (MBC) by the fumigation
extraction method (Vance et al. 1987). Triplicate soil samples
(30 g moist soil) were extracted with 2 M KCl, and inorganic N
contentwas determined colourimetrically using amodification of
Miranda et al. (2001) for NO3
 (plus NO2
) and Forster (1995)
for NH4
+. A 50-g subsample of soil from both soil zones was
taken for determination of gravimetric moisture content after
drying at 105C for 48 h.
Calculations and statistical analysis
Plant and soils data were analysed using the General Linear
Model (see exceptions below) in SAS (version 8.02, SAS
Institute, Cary, NC, USA). Where significant effects were
observed, pair-wise comparisons were made using Tukey?s
test (Zar 1999). Mycorrhizal colonisation data were arcsine
transformed (Zar1999)before analysis, and arepresented as such.
Mean hourly respiration data were used to calculate CO2
respired each day, and thence, cumulative respiration (CR) on
day Y was calculated following Eqn 2.
CR
Y
? S?X
DAY1
? X
DAY2
. . . ? X
DAYY
?; ?2?
where X= the total CO2 respired on a given day.
Soil respiration data were analysed as repeated-measures
using the ANOVA procedure in SAS. All respiration data are
presented starting from day 16 as data before this were highly
variable due to the small size of the plants and technical problems
with the gas handling system.
Results
Soil respiration
Cumulative respiration increased linearly over the course of the
experiment (Fig. 1). Total respiration of rhizocosms in soil
without plants was ~30% of that in rhizocosms containing
230 Functional Plant Biology T. R. Cavagnaro et al.
plants. In rhizocosms containing soil only, cumulative respiration
increased significantly with time (P < 0.0001) and was lower in
the high P addition treatment (P = 0.03). In these rhizocosms,
daily means of hourly respiration rates showed a significant
interaction between time and P addition treatment (P= 0.01),
explained by a gradual decrease in the difference between the
two P levels with time (Fig. 2).
In rhizocosms containing plants, cumulative respiration
increased significantly with time (P < 0.0001), but there were
no differences between genotypes or P addition treatments
(Fig. 1). This was also true for daily means of hourly
respiration (Fig. 2). Spikes in soil CO2 emissions closely
followed watering of pots (arrowed in Fig. 2). The amount of
CO2 respired per hour (averaged over the last 5 days of the
experiment; see Fig. 2) per unit root dry weight at harvest (see
below; Fig. 3c) was significantly (P = 0.0138) higher in the 76R
MYC+ plants than rmc plants, irrespective of P addition
treatment.
0
0.4
0.8
1.2
1.6
2
16 20 24 28 32 36 40 44 48 52 56 60 64 68 72 76 80
Time (days)D
ai
ly
 m
ea
ns
 o
f h
ou
rly
 re
sp
ira
tio
n 
(m
g C
-C
O 2
/h
r)
Soil Low P
Soil High P
76R MYC+ Low P
76R MYC+ High P
rmc  High P
rmc  Low P
?
? ?
?
?
?
?
?
?
?
?
?
?
?
Fig. 2. Daily mean of hourly respiration (mg C-CO2/h) from rhizocosms.
Treatments were: soil only/low P, soil only/high P, rmc/low P, rmc/high P,
76R MYC+/low P, and 76R MYC+/high P. Low P and High P refer to P1.5
and P7.5 treatments, respectively, i.e. 1.5 and 7.5 mg P added/g soil. Arrows
indicate time of watering. Values are mean 
 s.e., n= 6.
0
200
400
600
800
1000
1200
1400
16 20 24 28 32 36 40 44 48 52 56 60 64 68 72 76 80
Time (days)
Cu
m
ul
at
iv
e 
re
sp
ira
tio
n 
(m
g C
-C
O 2
)
Soil Low P
Soil High P
76R MYC+ Low P
76R MYC+ High P
rmc  High P
rmc  Low P
Fig. 1. Cumulative CO2 respired over the course of the experiment
(mg C-CO2) in rhizocosms. Treatments were: soil only/low P, soil only/
high P, rmc/low P, rmc/high P, 76R MYC+/low P, and 76R MYC+/high P.
Low P and High P refer to P1.5 and P7.5 treatments, respectively, i.e. 1.5 and
7.5mg P added/g soil. Values are mean 
 s.e., n= 6.
4.8
4.6
P1.5 P7.5 P1.5 P7.5
P1.5 P7.5 P1.5 P7.5 P1.5 P7.5 P1.5 P7.5
Sh
oo
t d
ry
 w
ei
gh
t (
g)
R
oo
t d
ry
 w
ei
gh
t (
g)
76R MYC+ rmc
R
oo
t  
le
ng
th
 d
en
sit
y 
(cm
 g?
1 ) 76R MYC+ rmc
B
A
76R MYC+
rmc
B
A
A
M
F 
co
lo
ni
sa
tio
n
76R MYC+
rmc
A
B
4.4
4.2
4.0
5.0
4.0
3.0
2.0
1.0
0.0
0.30
0.25
0.20
0.15
0.10
0.05
0.00
2.5
2.0
1.5
1.0
0.5
0.0
P1.5 P7.5 P1.5 P7.5
(a)
(c) (d)
(b)
Fig. 3. (a) Shoot dry weight (g), (b) root length density (cm g1 dry soil), (c) root dry weight (g), and
(d) mycorrhizal colonisation at harvest (percent arcsine transformed), of 76R MYC+ and rmc tomato
plants grown in the low (P1.5) and high (P7.5) P addition treatments, respectively, i.e. 1.5 and 7.5 mg P
added/g soil.Values aremean 
 s.e.,n=6.Different letters indicate significant differences atP< 0.05 for
genotype main effect.
The mycorrhizal contribution to soil respiration Functional Plant Biology 231
Soil microbial biomass, PLFA and mineral N pools
Microbial biomass C was not influenced by the P addition (low
or high) or plant (76R MYC+, rmc, soil only) treatments, alone
or in combination (Tables 2, 3). The total PLFA was not
different between treatments (P < 0.6), with a mean (
s.e.)
value of 2045 (
317) ng/g soil. Based on multivariate
statistical analysis of PLFA profiles, soil microbial
communities did not differ consistently between any of the
low P experimental treatments (data not shown). This analysis
included the 29 PLFA that were present in more than 10%
of samples.
Soil NO3
 concentrations (Tables 2, 3) were higher in the
soil blanks (5.8mgN/g soil) than in the treatments with plants
(0.02mgN/g soil). There were no differences in soil NH4
+
concentrations (Tables 2, 3), which were 0.7mgN/g soil in
response to any of the experimental treatments.
Plant growth, nutrient uptake and mycorrhizal colonisation
Shoot dry weights of 76R MYC+ and rmc plants did not differ
at harvest (Fig. 3a), whereas root length density (Fig. 3b) and
root dry weights (Fig. 3c) were higher for rmc plants. Total
biomass (shoots + roots) did not differ between genotypes or
P addition treatments (data not shown). AMF colonised both
the epidermal and cortical cell layers of the 76R MYC+
roots (~20% root length colonised), whereas colonisation of
rmc was restricted to the root epidermis and remained low
(less than 1%) (Fig. 3d).
Shoot concentrations of Zn (Fig. 4a) and P (Fig. 4b) were
58 and 20% higher in 76R MYC+ plants than rmc plants,
respectively. There were no significant differences in plant B,
Ca, Fe, Mg, N, Na and S concentrations (data not shown).
Discussion
Colonisation of roots by AMF did not have a measurable effect
on cumulative or daily means of hourly soil respiration. Some
earlier studies report an increase in respiration in roots colonised
by AMF (Pang and Paul 1980; Peng et al. 1993), but others do
not (Silsbury et al. 1983). Size asymmetry between mycorrhizal
and non-mycorrhizal plants is an important factor. For example,
using the same automated respiration monitoring system,
Langley et al. (2005) reported higher respiration from
Helianthus annuus L. plants colonised by AMF, an effect
attributed to higher plant growth in the mycorrhizal treatments.
Although no net effect of AMF on soil respiration was detected
in this study, there may have been differences in root or
mycorrhizal processes. Despite similar shoot and total plant
biomass, root biomass and root length density were higher in
rmc plants, which had essentially no (<1%) mycorrhizal
colonisation. The specific root length of the 76R MYC+ roots
was significantly higher (12.9
 0.3 cm/mg dry weight) than in
the rmc plants (10.8
 0.7 cm/mg dry weight). Thus, the sum of
root +AMF respiration (both autotrophic and heterotrophic
Table 2. Soil microbial biomass and mineral N pools
Microbial biomass C (MBC), NH4
+-N concentration (mg/g) and NO3
-N concentration (mg/g soil) in the surface 0--15 cm, n= 6
MYCA P1.5 MYC P7.5 rmc P1.5 rmc P7.5 Soil P1.5 Soil P7.5
Mean s.e. Mean s.e. Mean s.e. Mean s.e. Mean s.e. Mean s.e.
MBC (mg/g) 191.5 24.2 197.1 18.9 159.1 17.4 173.7 8.6 219.4 21.5 198.7 24.9
NH4
+-N (mg/g) 0.6 0.1 0.7 0.1 0.7 0.1 0.7 0.1 0.4 0.1 0.6 0.2
NO3
---N (mg/g) 0.02 0.01 0 0 0.02 0.02 0.02 0.02 6.3 0.6 5.4 0.7
AMYC=76R MYC+.
Table 3. Soil microbial biomass and mineral N pools ANOVA
GLM results given: n.s., not significant at P< 0.05, n= 6
Plant P Level PlantP level Block
MBC n.s. n.s. n.s. n.s.
NH4
+-N n.s. n.s. n.s. 0.0006
NO3
-N <0.0001 n.s. n.s. n.s.
0
10
20
30
40
50
60
P1.5 P7.5 P1.5 P7.5
Sh
oo
t Z
n 
co
nc
. (?
g 
g?
1 ) 
76R MYC+
rmc
B
A
4000
4500
5000
5500
6000
6500
7000
P1.5 P7.5 P1.5 P7.5
Sh
oo
t P
 c
on
c.
 (?
g 
g?
1 ) 
76R MYC+
rmc
B
A
(a) (b)
Fig. 4. (a) Shoot Zn concentration, and (b) shoot P concentration, of 76R MYC+ and rmc tomato plants
grown in the low (P1.5) and high (P7.5) P addition treatments, respectively, i.e. 1.5 and 7.5 mg P added/g soil.
Values are mean
 s.e., n = 6. Different letters indicate significant differences at P< 0.05 for genotype main
effect.
232 Functional Plant Biology T. R. Cavagnaro et al.
respiration) in the 76R MYC+ treatment may have been
equivalent to that of the more abundant, thicker but
uncolonised roots in the rmc treatment. A lack of significant
difference in cumulative respiration per unit root length
density at the end of the experiment (data not shown) further
supports the conclusion that the two genotypes did not differ in
terms of net effects on soil respiration. Importantly, hourly
respiration (average over the last 5 days of the experiment)
per unit dry weight (at harvest) was higher in the 76R MYC+
(0.44
 0.05mg C/g RDW) than rmc plants (0.27
 0.04mg C/g
RDW) (also compare Figs 2, 3c), indicating a significant effect
of AMF colonisation on root respiration (see also Mortimer et al.
2005).
Mycorrhizal plants achieved higher uptake of P and Zn with
similar soil respiration as non-mycorrhizal plants. There were
no differences in tissue N concentrations here (shoot N <1%
DW), although AMF can improve the N nutrition of plants
(Marschner and Dell 1994; Ryan and Ash 1999; Cavagnaro et al.
2006). Although allocation of C to AMF can be <20% of a
plant?s photoassimilates (Jakobsen and Rosendahl 1990), these
plants were not highly colonised (<20%), implying that these
mycorrhizal plants may have allocated less C to AMF. The
allocation of C to AMF may be approximately equal to
the difference in root biomass C that was observed between
the genotypes. Since the plant tissue contained 0.4 g C/g biomass
(based on values for shoots and stems), then it follows that ~0.2 g
more root C was present in the non-mycorrhizal plants. Based
upon this value, we estimate that ~9%more Cwas present as root
biomass in the non-mycorrhizal plants, given a total of
2.3 g C/plant in both genotypes. This simple estimation of C
allocation to AMF, along with the finding that soil respiration is
slightly higher per unit root biomass in mycorrhizal plants, and
the assumption that AMF received <20% of photoassmilates
(Jakobsen and Rosendahl 1990), suggests that approximately
similar amounts of C may have been expended belowground in
both genotypes. If so, then there was higher nutrient uptake
per amount of C invested belowground, for construction,
maintenance and functioning of AMF (e.g. see Wright et al.
1998) in the mycorrhizal plants. These calculations are
indicative only, with more detailed modelling required before
generalisations can be made. To this end, further studies
of the type we report here, using soils with similarly low C
levels, so as to minimise ?background? heterotrophic respiration,
are required.
It is unlikely that differences in the wider soil biota affected
soil respiration in the mycorrhizal and non-mycorrhizal
treatments, mainly because MBC and total PLFA were similar
in all treatments. This is not unexpected given that microbial
biomass and total PLFA have been found to be highly correlated
with each other (Potthoff et al. 2006). As in a previous study,
PLFA analysis showed little difference in soil microbial
communities between the two tomato genotypes (Cavagnaro
et al. 2006). In that situation, the soil was from an organically-
managed farm, and had higher MBC and the number of PLFA
was also greater; 45 PLFA were present in >10% of the samples
v. 29 PLFA in this study. Thus, the lack of effect was consistent
across quite different soils. Using a DNA-based approach, shifts
in the total bacterial community composition (Marschner and
Timonen 2005), but not of the ammonia oxidising bacteria
(Cavagnaro et al. 2007a), have been observed in the
rhizosphere of these tomato genotypes and attributed to a
complex series of interactions between genotype, AMF species
and light levels.
In the absence of plants, the higher level of P addition to
this soil resulted in lower cumulative soil respiration. A similar
trend was observed in the treatments with plants, but was not
significant. These results are difficult to explain but may be
related to the availability of other ions. Phosphate can react
with cations to give soluble molecular and ionic species as
well as insoluble salts that may have affected microbial
activity (Robertson and Alexander 1992). Previous studies
have shown higher levels of soil respiration under low P
conditions, probably due to increased C allocation to root
production and activity (Keith et al. 1997; Nielsen et al.
1998), but at P concentrations much lower than this study.
The levels of extractable soil P in this study are considered
below excessive levels for vegetables (Maynard and Hochmuth
1997). The leaf tissue levels were in the adequate to high range
for this stage of growth in tomato, and the plants showed no
visual symptoms of P toxicity (Maynard and Hochmuth 1997).
Zinc levelswere also in the adequate to high range, also indicating
that P availability was not excessive, since overly high P levels
can induce Zn deficiency in plants (Marschner 1995). High soil
P can lead to a reduction in colonisation by AMF (Oliver et al.
1983; Baon et al. 1992), due to a reduction in fungal growth
and/or to increases in the growth of roots (Smith 1982; Thomson
et al. 1991; Bruce et al. 1994). Such effects were not seen here,
or in a survey of organic tomato farms in California, where there
was no correlation between mycorrhizal colonisation and
extractable P (L. E. Jackson and L. A. Saxe, pers. comm.).
Although plant available soil P was relatively high, it was
considerably lower than in other studies where functional
AMF were observed (e.g. Blanke et al. 2005). Further insight
may be gained in future studies by taking into account effects of
P on themetabolic activity of AMF (Ezawa et al. 2004; vanAarle
et al. 2005; Valentine and Kleinert 2007), extra-radicle growth
(Olsson et al. 2002; Cavagnaro et al. 2005) and the abundance of
arbuscules or other structures (Cavagnaro et al. 2001; Jackson
et al. 2002; Cavagnaro et al. 2003).
Conclusions
The formation of AM presents a complex series of tradeoffs
between the C cost of the fungi and the benefits of enhanced
nutrient supply to theplant (Johnson et al. 1997;Fitter et al. 2000).
When colonised by AMF, a plant?s nutrient demands can be
met via the mycorrhizal pathway (Smith et al. 2004), often
coupled with relatively lower root biomass (Cavagnaro et al.
2007b). That net soil CO2 respired did not differ between the rmc
and 76R MYC+ genotypes in this study, despite differences in
root length and biomass between mycorrhizal treatments,
indicates a trade-off between respiration associated with
mycorrhizal fungi and that required to support non-
mycorrhizal root function. Importantly, hourly respiration at
the end of the experiment per unit root dry weight was higher
in the 76R MYC+ than rmc plants. The genotypic approach to
control for AM colonisation helped avoid underestimation of
the heterotrophic component of soil respiration, and hence,
The mycorrhizal contribution to soil respiration Functional Plant Biology 233
potential overestimation of the contribution of AMF to soil
respiration. These data suggest that although net soil
respiration may not differ between plants colonised by AMF
and those not colonised, the mycorrhizal contribution to soil
respiration may indeed be significant, and yet the nutrient gains
per unit C expended belowground appears to be greater than in
non-mycorrhizal plants.
Acknowledgements
We are especially grateful to Sally Smith (University of Adelaide) and
Susan Barker (University of Western Australia), for allowing our
continued use of the rmc tomato mutant/wild-type system. We also thank
Katherine Sides (NAU) for her excellent technical assistance and that from
various members of the Jackson Laboratory (UC Davis). Thanks also to
Kate Scow for PLFA extraction and identification at UCDavis. The efforts of
two anonymous reviewers are also appreciated. The research was funded by
the California Department of Food andAgriculture Speciality Crops Program
(SA6674), the United States Department of Agriculture National Research
Initiative Soils and Soil Biology Program (2004--03329) and a National
Science Foundation grant to Nancy Johnson and George Koch
(DEB:9806529).
References
Bakhtiar Y, Miller D, Cavagnaro TR, Smith SE (2001) Interactions
between two arbuscular mycorrhizal fungi and fungivorous nematodes
and control of the nematode with fenamifos. Applied Soil Ecology 17,
107--117. doi: 10.1016/S0929-1393(01)00129-9
Baon JB, Smith SE, Alston AM, Wheeler RD (1992) Phosphorus
efficiency of three cereals as related to indigenous mycorrhizal
infection. Australian Journal of Agricultural Research 43, 479--491.
doi: 10.1071/AR9920479
Barker SJ, Stummer B, Gao L, Dispain I, O?Connor PJ, Smith SE (1998)
A mutant in Lycopersicon esculentum Mill. with highly reduced VA
mycorrhizal colonization, isolation and preliminary characterisation.
The Plant Journal 15, 791--797. doi: 10.1046/j.1365-313X.1998.
00252.x
Blanke V, Renker C, Wagner M, Fu?llner K, Held M, Kuhn AJ, Buscot F
(2005) Nitrogen supply affects arbuscular mycorrhizal colonization of
Artemisia vulgaris in a phosphate-polluted field site. New Phytologist
166, 981--992. doi: 10.1111/j.1469-8137.2005.01374.x
Bligh EG, Dyer WM (1959) A rapid method of lipid extraction and
purification. Canadian Journal of Biochemistry and Physiology 35,
911--917.
Bossio DA, Scow KM (1998) Impacts of carbon and flooding on soil
microbial communities, phospholipid fatty acid profiles and substrate
utilization patterns. Microbial Ecology 35, 265--278. doi: 10.1007/
s002489900082
Bruce A, Smith SE, Tester M (1994) The development of mycorrhizal
infection in cucumber: effects of P supply on root growth, formation
of entry points and growth of infection units. New Phytologist 127,
507--514. doi: 10.1111/j.1469-8137.1994.tb03968.x
Butler JL, Williams MA, Bottomley PJ, Myrold DD (2003)
Microbial community dynamics associated with rhizosphere
carbon flow. Applied and Environmental Microbiology 69,
6793--6800. doi: 10.1128/AEM.69.11.6793-6800.2003
Cavagnaro TR, Smith FA, Lorimer MF, Haskard KA, Ayling SM, Smith SE
(2001) Quantitative development of Paris-type arbuscular mycorrhizas
formed between Asphodelus fistulosus and Glomus coronatum.
New Phytologist 149, 105--113. doi: 10.1046/j.1469-8137.2001.00001.x
Cavagnaro TR, Smith FA, Ayling SM, Smith SE (2003) Growth and
phosphorus nutrition of a Paris-type arbuscular mycorrhizal
symbiosis. New Phytologist 157, 127--134. doi: 10.1046/j.1469-8137.
2003.00654.x
Cavagnaro TR, Smith FA, Hay G, Carne-Cavagnaro VL, Smith SE
(2004) Inoculum type does not affect overall resistance of an
arbuscular mycorrhiza-defective tomato mutant to colonisation but
inoculation does change competitive interactions with wild-type
tomato. New Phytologist 161, 485--494. doi: 10.1111/j.1469-8137.
2004.00967.x
Cavagnaro TR, Smith FA, Smith SE, Jakobsen I (2005) Functional diversity
in arbuscular mycorrhizas: exploitation of soil patches with different
phosphate enrichment differs among fungal species. Plant, Cell &
Environment 164, 485--491.
Cavagnaro TR, Jackson LE, Six J, Ferris H, Goyal S, Asami D, Scow KM
(2006) Arbuscular mycorrhizas, microbial communities, nutrient
availability, and soil aggregates in organic tomato production. Plant
and Soil 282, 209--225. doi: 10.1007/s11104-005-5847-7
Cavagnaro TR, Jackson LE, Scow KM, Hristova KR (2007a) Effects
of arbuscular mycorrhizas on ammonia oxidizing bacteria in an organic
farm soil. Microbial Ecology 54, 618--626. doi: 10.1007/s00248-007-
9212-7
Cavagnaro TR, Sokolow SK, Jackson LE (2007b) Mycorrhizal effects on
growth and nutrition of tomato under elevated atmospheric carbon
dioxide. Functional Plant Biology 34, 730--736. doi: 10.1071/
FP06340
Cheng W (1996) Measurement of rhizosphere respiration and organic
matter decomposition using natural 13C. Plant and Soil 183, 263--268.
doi: 10.1007/BF00011441
Coleman DC, Andrews R, Ellis JE, Singh JS (1976) Energy flow
and partition in selected managed and natural ecosystems.
Agro-Ecosystems 3, 45--54. doi: 10.1016/0304-3746(76)90099-8
Cramer MD, Lewis OAM, Lips SH (1993) Inorganic carbon fixation and
metabolism inmaize roots as affected by nitrate and ammoniumnutrition.
Physiologia Plantarum 89, 632--639. doi: 10.1111/j.1399-3054.1993.
tb05226.x
Ezawa T, Cavagnaro TR, Smith SE, Smith FA, Ohtomo R (2004) Rapid
accumulation of polyphosphate in extraradical hyphae of an arbuscular
mycorrhizal fungus as revealed by histochemistry and a polyphosphate
kinase/luciferase system. New Phytologist 161, 387--392. doi: 10.1046/j.
1469-8137.2003.00966.x
FitterAH,HeinemeyerA, StaddonPL (2000)The impact of elevatedCO2 and
global climate change on arbuscular mycorrhizas: a mycocentric
approach. New Phytologist 147, 179--187. doi: 10.1046/j.1469-8137.
2000.00680.x
Forster JC (1995) Soil nitrogen. In ?Methods in applied soil microbiology and
biochemistry?. (Eds K Alef, P Nannipiero) pp. 79--87. (Academic Press:
San Diego)
Gange AC (2000) Arbuscular mycorrhizal fungi, collembola and plant
growth. Trends in Ecology & Evolution 15, 369--372. doi: 10.1016/
S0169-5347(00)01940-6
Gao LL (2002) Control of arbuscular mycorrhizal colonisation. Studies of
a mycorrhiza-defective tomato mutant. PhD thesis, The University of
Adelaide, SA.
Guckert JB, Hood MA, White DC (1986) Phospholipid ester-linked fatty
acid profile changes during nutrient deprivation of Vibrio chlerae,
increase in the trans/cis ratio and proportions of cyclopropyl fatty
acids. Applied and Environmental Microbiology 52, 794--801.
Ingham RE, Trofymow JA, Ingham ER, Coleman DC (1985) Interactions of
bacteria, fungi, and their nematode grazers: effects on nutrient cycling and
plant growth. Ecological Monographs 55, 119--140. doi: 10.2307/
1942528
Jackson LE, Miller D, Smith SE (2002) Arbuscular mycorrhizal colonization
and growth of wild and cultivated lettuce in response to nitrogen and
phosphorus. Scientia Horticulturae 94, 205--218. doi: 10.1016/S0304-
4238(01)00341-7
Jakobsen I, Rosendahl IL (1990) Carbon flow into soil and external hyphae
from roots of mycorrhizal cucumber roots. New Phytologist 115, 77--83.
doi: 10.1111/j.1469-8137.1990.tb00924.x
234 Functional Plant Biology T. R. Cavagnaro et al.
Johnson NC, Graham JH, Smith FA (1997) Functioning of mycorrhizal
associations along the mutualism-parasitism continuum.
New Phytologist 135, 575--586. doi: 10.1046/j.1469-8137.1997.00729.x
Keith H, Jacobsen KL, Raison RJ (1997) Effects of soil phosphorus
availability, temperature and moisture on soil respiration in Eucalyptus
pauciflora forest. Plant and Soil 190, 127--141. doi: 10.1023/
A:1004279300622
Kra?mer S, Green GM (1999) Phosphorus pools in tree and intercanopy
microsites of a Juniper--Grass ecosystem. Soil Science Society
of America Journal 63, 1901--1905.
Kuzyakov Y, Domanski G (2002) Model for rhizodeposition and CO2
efflux from planted soil and its validation by 14C pulse labeling of
ryegrass. Plant and Soil 239, 87--102. doi: 10.1023/A:1014939120651
Langley JA, JohnsonNC,KochGW (2005)Mycorrhizal status influences the
rate but not the temperature sensitivity of soil respiration. Plant and Soil
277, 335--344. doi: 10.1007/s11104-005-7932-3
Luo Y, Jackson RB, Field CB, Mooney HA (1996) Elevated CO2 increases
belowground respiration in California grasslands. Oecologia 108,
130--137. doi: 10.1007/BF00333224
Marschner H (1995) ?Mineral nutrition of higher plants.? (Academic Press
Ltd: San Diego)
Marschner H, Dell B (1994) Nutrient uptake in mycorrhizal symbiosis.
Plant and Soil 159, 89--102.
Marschner P, Timonen S (2005) Interactions between plant species and
mycorrhizal colonization on the bacterial community composition in
the rhizosphere. Applied Soil Ecology 28, 23--36. doi: 10.1016/j.apsoil.
2004.06.007
MaynardDN,HochmuthGJ (1997) ?Knott?s handbok for vegetable growers.?
(John Wiley & Sons Inc.: New York)
Miranda KM, Espey MG, Wink DA (2001) A rapid, simple
spectrophotometric method for simultaneous detection of nitrate and
nitrite. Nitric Oxide 5, 62--71. doi: 10.1006/niox.2000.0319
Mortimer PE, Archer E, Valentine AJ (2005) Mycorrhizal C costs and
nutritional benefits in developing grapevines. Mycorrhiza 15, 159--165.
doi: 10.1007/s00572-004-0317-2
Nielsen KL, Bouma TJ, Lynch JP, Eissenstat DM (1998) Effects of
phosphorus availability and vesicular-arbuscular mycorrhizas on the
carbon budget of common bean (Phaseolus vulgaris). New Phytologist
139, 647--656. doi: 10.1046/j.1469-8137.1998.00242.x
Oliver AJ, Smith SE, Nicholas DJD, Wallace W, Smith FA (1983)
Activity of nitrate reductase in Trifolium subterraneum: effects of
mycorrhizal infection and phosphate nutrition. New Phytologist 94,
63--79. doi: 10.1111/j.1469-8137.1983.tb02722.x
Olsson PA, van Aarle IM, Allaway WG, Ashford AE, Rouhier H (2002)
Phosphorus effects on metabolic processes in monoxenic arbuscular
mycorrhiza cultures. Plant Physiology 130, 1162--1171. doi: 10.1104/
pp.009639
Pang PC, Paul EA (1980) Effects of vesicular arbuscular mycorrhiza on 14C
and 15N distribution in nodulated faba beans. Canadian Journal of Soil
Science 60, 241--250.
Peng S, Eissenstat DM, Graham JH, Williams K, Hodge NC (1993) Growth
depression in mycorrhizal citrus at high-phosphorus supply. Plant
Physiology 101, 1063--1071.
Potthoff M, Steenwerth KL, Jackson LE, Drenovsky RE, Scow KM,
Joergensen RG (2006) Soil microbial community composition as
affected by restoration practices in California grassland. Soil Biology &
Biochemistry 38, 1851--1860. doi: 10.1016/j.soilbio.2005.12.009
Poulsen KH, Nagy R, Gao L-L, Smith SE, Bucher M, Smith FA, Jakobsen I
(2005) Physiological and molecular evidence for Pi uptake via the
symbiotic pathway in a reduced mycorrhizal colonization mutant in
tomato associated with a compatible fungus. New Phytologist 168,
445--454. doi: 10.1111/j.1469-8137.2005.01523.x
Rillig MC (2004) Arbuscular mycorrhizae and terrestrial ecosystem
processes. Ecology Letters 7, 740--754. doi: 10.1111/j.1461-0248.
2004.00620.x
Robertson BK, Alexander M (1992) Influence of calcium, iron, and pH on
phosphate availability for microbial mineralization of organic chemicals.
Applied and Environmental Microbiology 58, 38--41.
Rochette P, Angers DA (1999) Soil surface carbon dioxide fluxes induced by
spring, summer, and fallmoldboard plowing in a sandy loam. Soil Science
Society of America Journal 63, 621--628.
Ryan MH, Ash A (1999) Effects of phosphorus and nitrogen on growth of
pasture plants and VAM fungi in SE Australian soils with contrasting
fertiliser histories (conventional and biodynamic). Agriculture
Ecosystems and Environments 73, 51--62. doi: 10.1016/S0167-8809
(99)00014-6
Sah RN, Miller RO (1992) Spontaneous reaction for acid dissolution of
biological tissues in closed vessels. Analytical Chemistry 64, 230--233.
doi: 10.1021/ac00026a026
Silsbury JH, Smith SE, Oliver AJ (1983) A comparison of growth efficiency
and specific rate of dark respirationof uninfected and vesicular-arbuscular
mycorrhizal plants of Trifolium subterraneum L. New Phytologist 93,
555--566. doi: 10.1111/j.1469-8137.1983.tb02706.x
Smith SE (1982) Inflow of phosphate into mycorrhizal and non-mycorrhizal
plants of Trifolium subterraneum at different levels of soil phosphate.
NewPhytologist 90, 293--303. doi: 10.1111/j.1469-8137.1982.tb03261.x
Smith SE, Read DJ (1997) ?Mycorrhizal symbiosis.? (Academic Press Ltd.
Cambridge)
Smith SE, Smith FA, Jakobsen I (2004) Functional diversity in arbuscular
mycorrhizal (AM) symbioses: the contribution of the mycorrhizal P
uptake pathway is not correlation with mycorrhizal reponses in growth
or total P uptake. New Phytologist 162, 511--524. doi: 10.1111/j.1469-
8137.2004.01039.x
Tennant D (1975) A test of the modified line intersect method of estimating
root length. Journal of Ecology 63, 995--1001.
Thomson BD, Robson AD, Abbott LK (1991) Soil mediated effects
of phosphorus supply on the formation of mycorrhizas by
Scutellospora calospora (Nicol. & Gerd.) Walker and Sanders on
subterranean clover New Phytologist 118, 463--469. doi: 10.1111/j.
1469-8137.1991.tb00028.x
Valentine AJ, Kleinert A (2007) Phosphate deficiency affects respiratory
metabolism of dark CO2 fixation in mycorrhizal roots. Mycorrhiza 17,
137--143. doi: 10.1007/s00572-006-0093-2
van Aarle IM, Cavagnaro TR, Smith SE, Smith FA, Dickson S (2005)
Metabolic activity of Glomus intraradices in Arum- and Paris-type
arbuscular mycorrhizal colonization. New Phytologist 166, 611--618.
doi: 10.1111/j.1469-8137.2005.01340.x
Vance ED, Brookes PD, Jenkinson DS (1987) An extraction method to
estimate soil microbial biomass C. Soil Biology & Biochemistry 19,
703--707. doi: 10.1016/0038-0717(87)90052-6
Wright DP, Scholes JD, ReadDJ (1998)Mycorrhizal sink strength influences
whole-plant carbon balance of Trifolium repens L. Plant, Cell &
Environment 21, 881--891. doi: 10.1046/j.1365-3040.1998.00351.x
Zar JH (1999) ?Biostatistical analysis.? (Prentice Hall: Upper Saddle
River, NJ)
Manuscript received 26 November 2007, accepted 13 March 2008
The mycorrhizal contribution to soil respiration Functional Plant Biology 235
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