Intraspecific transfer of carbon between plants linked by a common mycorrhizal network

Abstract

To quantify the involvement of arbuscular mycorrhiza (AM) fungi in the intraspecific transport of carbon (C) between plants we fumigated established Festuca ovina turf for one week with air containing depleted 13C. This labelled current assimilate in a section of mycorrhizal or non-mycorrhizal turf. Changes in the 13/12C ratio of adjacent, unfumigated plants, therefore, allowed the movement of C between labelled and unlabelled plants to be estimated. In mycorrhizal turves, 41% of the C exported to the roots from the leaves was transported to neighbouring plants. The most likely explanation of this is was the transport of C via a common hyphal network connecting the roots of different plants. No inter-plant transport of C was detected in non-mycorrhizal turves. There was no evidence that the C left fungal structures and entered the roots of receiver plants. Mycorrhizal colonisation increased carbon transport from leaves to root from 10% of fixed carbon when non-mycorrhizal to 36% in mycorrhizal turves. These results suggest that AM fungi impose a significantly greater C drain on host plants than was previously thought.

Full text

Plant and Soil 192: 153–159, 1997. 153
c
1997 Kluwer Academic Publishers. Printed in the Netherlands.
Intraspecific transfer of carbon between plants linked by a common
mycorrhizal network
J.D. Graves
1
, N.K. Watkins
1
, A.H. Fitter
1
, D. Robinson
2
and C. Scrimgeour
2
1
Biology Department, The University of York, PO Box 373, York YO1 5YW, UK and
2
Scottish Crop Research
Institute, Invergowrie, Dundee DD2 5DA, UK
Received 10 December 1996. Accepted in revised form 7 May 1997
Keywords: arbuscular mycorrhiza,carbonallocation, carbon cost, carbon transport, commonmycorrhizalnetwork,
13
C
Abstract
To quantify the involvement of arbuscular mycorrhiza (AM) fungi in the intraspecific transport of carbon (C)
between plants we fumigated established Festuca ovina turf for one week with air containing depleted
13
C. This
labelled current assimilate in a section of mycorrhizal or non-mycorrhizal turf. Changes in the
13 12
C ratio of
adjacent, unfumigated plants, therefore, allowed the movement of C between labelled and unlabelled plants to be
estimated.Inmycorrhizalturves,41%oftheCexportedtotherootsfromtheleaveswastransportedtoneighbouring
plants. The most likely explanation of this is was the transport of C via a common hyphal network connecting
the roots of different plants. No inter-plant transport of C was detected in non-mycorrhizal turves. There was no
evidencethat the Cleft fungalstructuresandenteredtheroots of receiver plants. Mycorrhizalcolonisationincreased
carbontransportfrom leavesto rootfrom10% of fixed carbon when non-mycorrhizalto 36% in mycorrhizalturves.
These results suggest that AM fungi impose a significantly greater C drain on host plants than was previously
thought.
Introduction
The hyphae of arbuscular mycorrhizal (AM) fungi
have been observed to link the roots of plants of the
samespecies (Hirrel and Gerdemann, 1979)and plants
of different species (Heap and Newman, 1980). AM
colonisation is believed frequently to arise from grow-
ing roots contacting the hyphae emanating from the
roots of other plants in the community. Therefore, it is
likely that most plants capable of infection are inter-
connected by a common mycorrhizal network (CMN)
(Newman, 1988) unless there is a greater degree of
specificity in the interaction than is generally believed.
Studiesusing
14
Chavedemonstratedcarbon move-
ment between plants linked by mycorrhizal hyphae
(Francis and Read, 1984; Hirrel and Gerdemann,
1979). However, such studies have been unable to:
(i) measure transport in both directions and thereby
demonstrate whether there is any net transport in one
FAX No:+441904432860.E-mail:JDG3@York.ac.uk
direction, since no simultaneous measurement of
12
C
flow is possible, and (ii) in the case of pulse labelled
experimentstoquantifytheC transfer,becausethespe-
cific activity of the source is unknown. Furthermore,
the factors determining movement of C are uncertain.
Francis and Read (1984) found that shading of the
receiver plant increased C movement towards it, sug-
gestingthatsource-sinkrelationshipscontrolledmove-
ment. In contrast, Waters and Borowicz (1994) found
that there was no increase in
14
C transfer when one
neighbour was clipped above ground to alter its sink
strength for C.
Some of the problems inherent in using
14
C
have been overcome by using ratios of stable iso-
topes (
13 12
C) to trace C transfer. Watkins et al.
(1996)utilised the natural difference in
13
C abundance
between the C
3
species Plantago lanceolata (mean
13
C-28‰) and the C
4
Cynodon dactylon (mean
13
C
-13.5‰) to estimate C transfer between them. They
demonstrated C transfer from the C
3
to the C
4
plant
JS: PIPS No. 141614 BIO2KAP
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154
was in the range 0 to 41% (i.e. up to 41% of the C
in the ‘receiver’ plant originated from the ‘donor’ to
whichit wasconnected).However,C transferwas usu-
ally less than 10%. Clearly a major limitation of this
technique is that it can only be used on C
3
-C
4
pairs
of plants. We describe here a
13
C technique which
can quantify C movement between plants with the
same photosynthetic pathway. It relies on a fumiga-
tion procedure whereby half the plants are exposed to
air from which all the CO
2
has been removed and then
replaced with CO
2
from a bottled gas source. The bot-
tled CO
2
is much more depleted in
13
C(
13
C -30‰)
than atmospheric CO
2
is ( -8‰) and can therefore be
used to isotopicallylabel plants exposed to it. We used
this technique to quantify AM mediated C movement
between Festuca ovina plants growing in a turf to esti-
mate how much C was transferred between plants of
the same species when their roots were colonised or
not by AM.
Material and methods
Turf preparation
Nine turves of Festuca ovina (25 cm 40 cm 14 cm
depth)werecutfromlarge(2.5m 2.5m 40cmdepth)
turveswhich had been grownon a 50%sand/50%John
Innes compost mixture in a glasshouse for over a year.
One of the large turves was non-mycorrhizal whilst
the other had been inoculated with 550 g of a mixture
of four species of the genus Glomus obtained from
Agricultural Genetics Company (MicroBio Division,
Rothamsted Experimental Station, Harpenden, UK).
Turves were watered as required and when there were
signs of nutrient deficiency they were watered with
a Rorison’s nutrient solution. Mycorrhizal and non-
mycorrhizalturves were of a similar appearance. Each
small turf was placed into a plastic box (25.5 cm 42
cm 15 cm and left for approximately 8 weeks before
use. A partition was placed across the middle of each
box and in orderto ensure that there was no subsurface
leakage of air between fumigation compartments this
partition penetrated 2 cm below the soil surface. The
turf was watered daily and to encourage plant growth,
two days prior to the fumigation period the turf was
watered with a Rorison’s solution modified to contain
no phosphorous.
Fumigation procedure
Each turf was fumigatedseparately for a period of one
week. In total five mycorrhizal turves were treated and
four non-mycorrhizal turves (one less than intended
owing to the breakdown of the growth chamber). For
each fumigation run a turf was placed within a Sanyo
Fitotron growth chamberat 16 C day/12 Cnighttem-
perature, with a photosynthetic photon flux density of
250 mol m
2
s
1
at plant height during the 16 hour
photoperiod. Two perspex boxes (25 cm 20 cm 20
cm height, volume 101) were placed over the turf, one
on either side of the central partition, and airtight seals
made at their bases (see Figure 1a). Air was drawn
througheach of the boxes by fans at a rate of 2.5 1 s
1
.
One side of the turf received air from the outside of
the building (containing CO
2
with
13
C = -8‰where
13
C=[(
13 12
C
sample
/
13 12
C
standard
)-1 1000). The oth-
er side of the turf received air from which all the
CO
2
had been removed by passing through a soda
lime column and replaced with CO
2
from a cylinder
(with
13
C<30‰; Figure 1). CO
2
concentration in the
airstream was adjusted to that in the ambient air using
an ADC WA524 (Analytical Development Company,
Hoddesdon, UK) CO
2
controller.
Plant sampling and analysis
Samples were taken from each corner of each com-
partment (see Figure 1b). Thus in each compartment,
two samples were taken from a point 2 cm from the
divide and two samples 18 cm away from the divide.
Several individual plants were removed in each sam-
ple and divided into roots and shoots. Only roots still
connected to the shoots were included in the root frac-
tion.
13
C was determined in oven dried samples as
described by Watkins et al. (1996). Analytical preci-
sion was 0.2‰. For each turfthepresenceor absence
of mycorrhizal colonisation was checked in root sub-
samples using the staining procedure of Merryweath-
er and Fitter (1991). All root samples from the myc-
orrhizal turves were heavily colonised by AM fun-
gi whilst all root samples from the non-mycorrhizal
turves were uncolonised.
Calculation of carbon transfer
In order to calculated whether C had been transferred
from plants rooted in the labelled side of the turf to
plants in the unlabelled side, comparisons were made
between plants close to the divide and those distant
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155
Figure 1. a.Plan of the system used to fumigate one side of a Festuca ovina turf with
13
C depleted CO
2
whilst the other side is flushed with
ambient air. b. Location and coding of tissue samples taken from the Festuca ovina turf after one weeks fumigation (top view). c. Root and
shoot
13
C values (mean and standard error) after one week of labelling.
plso6702.tex; 1/10/1997; 19:37; v.7; p.3

156
from the divide. With reference to Figure 1b we can
usethe followingsymbolsto designatethe
13
C values
of the four root samples:
Ab
r
= Ambient air, plant close to the border, root
sample
Ad
r
= Ambient air, plant distant from the border,
root sample
Fb
r
= fumigated with bottled CO
2
, plant close to
the border, root sample
Fd
r
= Fumigated with bottled CO
2
, plant distant
from the border, root sample
Shoot samples are designated by a subscript s
insteadofsubscriptr.Cin, ororiginatingfrom,labelled
plants was more depleted in
13
C(i.e.hadamore
negative
13
C) than C, in or originating from, plants
exposed to ambient CO
2
.
Ad and Fd values were considered to represent
‘background’
13
C abundances in the shoots and roots.
This assumed that plants close to the partition had con-
siderablymoremycorrhizalconnectionswith eachoth-
er than with those distant from the partition. So, Ad
plants were less likely to have received C from plants
grown in labelled CO
2
than Ab plant. Thus, Ab
r
-Ad
r
canbe used as an indexofhowmuchCmovedfromFb
to Ab plants during the week long fumigation. Had no
C moved this index would be zero. Had net movement
occurredfrom the plants receiving
13
C depleted C, this
indexwouldbenegative(Ab
r
<Ad
r
).Forthemovement
of C in the reverse direction, the corresponding index
isFb
r
-Fd
r
. Again,thiswouldbezero fornoC transfer,
but positive if C had been transferred.
The proportion of Carbon transferred can be esti-
mated in the mycorrhizal turves.
If index of
13
C
depleted C crossing the divide
and index of the background amount
of
13
C-depleted C received by the fumigated roots
then proportion of total
13
C-depleted C that is transferred
Table 1. Comparison of the index of carbon movement between myc-
orrhizal and non-mycorrhizal turves. Ab
r
-Ad
r
= a measure of the
13
C
depleted carbon that has moved from the fumigated side of the chamber
whilst Fb
r
-Fd
r
is a measure of the amount of
13
C enriched carbon that has
moved in the opposite direction (see text for details). values calculated
by one way analysis of variance, F
1 7
. The standard error of the mean is
shown in brackets
Mycorrhizal turves Non-mycorrhizal turves =
Ab -Ad -0.388 (0.152) 0.093 (0.107) 0.045
Fb -Fd 0.012 (0.234) 0.0425 (0.082) 0.92
The total amount of
13
C-depleted C absorbed (L
t
) can
be represented as:
Thus, the proportion of that
13
C-depleted C that is
transferred from shoots to roots (L
r s
) is given by:
This assumes that above and below ground biomass
are equal.
Results
Ab
r
-Ad
r
was negative in mycorrhizal turves, indicat-
ing net movement of C from labelled plants (Figure
1c, Table 1). This index was not significantly different
from zero in the non-mycorrhizal turves and was less
thanthe precisionofthe
13
Canalysis(see above).The
difference in Ab
r
-Ad
r
between mycorrhizal and non-
mycorrhizal turves was significant ( =0.045).Howev-
er, the index Fb
r
-Fd
r
was not significantly different
fromzeroineitherthe mycorrhizalor non-mycorrhizal
turves and, again, was less than analytical precision.
The proportion of C transferred in mycorrhizal
turves was 0.41 ( 0.04 SE). No detectable amount
of C was transferred in non-mycorrhizal turves since
no significant movements of C were detected (Figure
1c, Table 1).
Sinceeachturfwas fumigatedseparately,therewas
variationintheamountof Cabsorbedby theturvesas a
consequence of differentphotosyntheticrates between
individual exposures.
Therefore, the amount of
13
C-depleted C received
by the fumigated side of the turves will vary between
runs. However, there was a significant correlation
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157
Figure 2. The relationship between the indices of the amount of
13
C
depleted carbon reaching theroots offumigated plants (Fd
r
-Ad
r
)and
the amount of
13
C depleted carbon crossing the central divide into
the roots of plants treated with ambient air (Ab
r
-Ad
r
) in mycorrhizal
turves. Regression equation y=0.551x + 0.173, r
2
=0.92, F
1 3
=33.7,
=0.01. Each point represents one turf.
( =0.01) between the amount of
13
C depleted C that
was transferred (Ab
r
Ad
r
) and the total amount of
13
C
depleted C that was absorbed (Fd
r
-Ad
r
) (Figure 2).
Itispossiblethat gas leakageoffumigatedaircould
occurfromonehalfofthechambertotheother.If sothe
Ab plants would be closer to any contamination which
could alter their
13
C signal and such an alteration
would be apparent in shoot tissue. However, Ab
s
-Ad
s
was not significantly different from zero (Student’s t
test =0.27).
The proportion of that
13
C-depleted C that is trans-
ferred from shoots to roots(L
r s
) was 0.36 ( 0.07 SE)
in mycorrhizal turves compared with 0.10 ( 0.04 SE)
forthe non-mycorrhizalturves; the differencebetween
them was significant (Anova, F
1 7
=9.78, =0.017).
Discussion
Robustness of the technique
We have demonstrated that stable carbon measure-
ments coupled with fumigation using
13
C depleted
CO
2
can be usedto quantifyC transfer between plants.
This technique has several advantages over the use of
14
C especially where long term fumigation is need-
ed to measure processes near equilibrium. Using
13
C
labelling,net C transport over long periods of time can
be measured. At the same time, C transfer from shoots
to roots can be estimated. Experiments which use con-
tinuouslabelling with
14
Care capable of quantifyingC
flux from the labelled plant to the unlabelled plant but
are incapableof simultaneouslymeasuring the amount
of C moving in the opposite direction. Therefore the
net C transfer cannot be quantified. This severely con-
strainsthe designof experimentsto investigatethe fac-
tors that control C movement. This constraint which
can be relieved adopting the use of stable C isotope
labelling. A disadvantage of using
13
C labelling is that
the plants must be fumigated for a relatively long peri-
od of time. Judging from our results one week is near
the minimum period required to ensure a sufficient
change in the
13
C/
12
C ratio. In contrast
14
C labelling
can be used to investigate C movement over much
shorter time scales.
Potentially this technique can be used to measure
bi-directionalmovementofC,althoughweonlydetect-
ed transfer away from the fumigated plants. However,
the sensitivity of this technique is less for the plants
on the fumigated side of the divide as there is an addi-
tional source of error affecting their
13
C value. The
13
C value of the roots of these plants can be affect-
ed both by variation in the amount of
13
C-depleted C
absorbedphotosyntheticallyby thesampledplants and
by the amount of transport from neighbouring unfu-
migated plants. In contrast the
13
C value of plants on
the unfumigated side is only affected by the amount
of
13
C-depleted C transferred from the fumigated side
of the divide. We do not therefore believe that these
results demonstrate unequivocally one-way transfer.
A potentially serious problem with this technique
is the possible leakage of gas from one chamber to the
other. Thoseplantsnearestthesourceof contamination
would fix more of this gas thereby leading to a differ-
ence in the
13
C value of the Ab
s
and Ad
s
samples.
Two pieces of evidence indicate that such contami-
nation did not occur. First the amount transferred is
positively correlated with the amount of
13
C depleted
C entering the roots of the fumigated plants. Secondly,
if there were gaseous contamination then the shoots of
the unfumigatedplants would be more heavily labelled
than their roots and there would be a significant dif-
ference between the Ab
s
and Ad
s
samples. No such
difference was observed.
Role of C demand in plant-mycorrhizal associations
Being mycorrhizal increased carbon transfer to the
roots from 10% of the carbon fixed to 36%. This is
larger than other estimates of increased root allocation
in mycorrhizalplants; an increase of 10% in Vicia faba
(Pang and Paul, 1980), 14% in Allium porrum (Snell-
plso6702.tex; 1/10/1997; 19:37; v.7; p.5

158
groveetal.,1982)and Glycine max (Harrisetal.,1985)
and 37% in Citrus volkameriana (Peng et al., 1993).
Then are three possible reasons for the higher figure
in our study: i) that AM colonisation was better estab-
lished in this > 1 year old, perennial turf compared
with annual crop species a few weeks old; ii) a com-
mon mycorrhizal network interlinking many plants is
greater sink for C than the mycelium associated with
a single plant; and iii) that the root fraction of mycor-
rhizal plants was lower than that of non-mycorrhizal
plants, which is commonly observed (Hayman and
Mosse, 1972; Smith, 1982).
There was a significant difference in the index of
transport (Ab
r
-Ad
r
) between the mycorrhizal and non-
mycorrhizal turves. However, there was also greater
transport of
13
C-depleted C to the roots in the myc-
orrhizal plants. Therefore, if transport via the CMN
occurred, two factors might have determined the size
of that transfer: alteration in the source-sink relation-
ships within the donor plant; and the amount of C
exported from that plant into the CMN.
Our results rule out an alternative explanation for
the significant difference in Ab
r
-Ad
r
. This is that some
other process, such as transfer and re-assimilation of
root exudates (Jones and Darrah, 1995) was responsi-
blefor the observedCtransfer,detectableinthemycor-
rhizal turves because of the greater amount of labelled
C in the roots of those plants. However, if that were
true, C would enter the rootcells via an uptake process
and C metabolic pathways. Some of that C would then
be transferred to the shoots. In our experiments, no
transfer of ‘donor’ C to ‘receiver’ shoots was detected
(i.e. Ab
s
-Ad
s
0). This is consistent with otherresults
(e.g. Francis and Read, 1984; Watkins et al., 1996). It
strongly suggests that C transferred from one plant to
anothervia aCMN remains in fungalstructures within
roots.
Approximately 41% of the
13
C depleted C export-
ed from leaves to roots of fumigated F. o vi na was
transported to nearby unfumigated plants. This is con-
siderably greater than the average transfer detected by
Watkins et al. (1996) between neighbouring plants of
different species. Plantago lanceolata and Cynodon
dactylon (< 10%). The Watkins et al. study differed
from the present one in two important ways: AM
colonisation in the Festuca turves was well estab-
lished whereas between P. lanceolata and C. dacty-
lon colonisation was only a few weeks old; and intra-
specific CMN links may be more abundant than inter-
specific links. Although the latter explanation is possi-
ble,Chiarelloetal.(1982)showedinafieldexperiment
that movement of materials between mycorrhizally-
linked plants in a field experiment was apparently
unconstrained by taxonomic relatedness. The more
likely explanation is therefore, that colonisation was
more intense in this mature turf.
Conclusions
Our results confirm the demonstration by Watkins et
al. (1996) that transfer of C between the root systems
of neighbouring plants connected by a CMN can be
considerable. They emphasise that the carbon cost of
mycorrhizal symbiosis to the plant can be large, and
possibly much greater in established vegetation than
has been proposed from experiments on single plants.
There has been much debate on the physiological and
ecologicalsignificanceofC transportbyaCMN(New-
man, 1988), but this new evidence strongly suggests
that C stays within fungal structures in the roots of
‘receiver’plants and that speculations about the ability
of adult plants to ‘foster’ juveniles, for example, are
probably unfounded. The large quantities of C mov-
ing from root system to root system within the fungal
mycelium suggest that transfer is a reflection of the
growth pattern of the fungus, rather than the operation
of symbiotic processes.
Acknowledgements
This work was funded by the Natural Environment
Research Council under the Terrestrial Initiative in
GlobalEnvironmentalResearch (TIGER)programme.
We are grateful to Dr RDP Cargeeg of the Agricultural
Genetics Company for the supply of the mycorrhizal
inoculum.
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