New Phytol. (1999), 142, 539–550
Importance of light and CO on the effects
#
of endomycorrhizal colonization on growth
and photosynthesis of potato plantlets
(Solanum tuberosum) in an in vitro tripartite
system
D. L O U C H E-T E S S A N D I E R", G. S A M S O N#,
C. H E R N A! N D E Z-S E B A S T I A' #†, P. C H A G V A R D I E F F"
   Y. D E S J A R D I N S#*
" CEA-DEVM, LaP, Cadarache, 13108 Saint-Paul-lez-durance, France
# Centre de Recheche en Horticulture, Pavillon de l’Envirotron, UniversiteT Laval,
QC, Canada
Received 7 September 1998 ; accepted 23 February 1999
     
A factorial analysis was conducted to investigate the effects of different levels of photosynthetic photon flux (PPF)
and CO concentration on the interactions between the vesicular–arbuscular endomycorrhizal fungus Glomus
#
intraradices and potato plantlets (Solanum tuberosum) cultured in an in vitro tripartite system. We observed that
CO enrichment from 350 to 10000 ppm stimulated root colonization by the fungus, and that this stimulation was
#
more pronounced under high PPF (300 µmol m−# s−") than low PPF (60 µmol m−# s−"). Consistent with these
observations, the effects of G. intraradices on dry matter production in potato plantlets were strongly dependent
on the CO and PPF levels during cultivation. There was no significant effect of the mycorrhizal fungus on dry
#
matter production at 350 ppm of CO . However, under the high CO concentration, mycorrhiza had opposite
#
#
effects on dry matter production depending on the PPF : a decrease (k21%) and a stimulation (j25%) of dry
matter production after 2 wk of growth under low and high PPF, respectively, were observed in presence of G.
intraradices relative to plantlets grown in its absence. Furthermore, in mycorrhizal plantlets grown under high
levels of both PPF and CO , the chlorophyll and carotenoid contents as well as the quantum yields of
#
photosynthetic electron transport and the photochemical quenching qP of the chlorophyll-a fluorescence
measured near the PPF during growth were all higher than in non-infected plantlets. Our results therefore indicate
that mycorrhizal G. intraradices can alleviate the down regulation of photosynthesis related to sink limitation, and
its effect on dry matter production is strongly dependent on the levels of CO and PPF during growth which
#
determine the balance between the photosynthetic carbon uptake by the plantlets and the carbon cost by the
fungus.
Key words : chlorophyll-a fluorescence, CO enrichment, Glomus intraradices, micropropagation, mycorrhiza, O
#
#
evolution.
          
Symbiotic arbuscular mycorrhizal fungi can confer
several benefits to their host plants such as, enhanced
uptake of phosphorus (P) and other nutrients (Finlay
et al., 1992 ; Pearson & Jackobson, 1993), resistance
*Author for correspondence (tel 418 656 2131, extn 2359 ; e-mail
Yves.Desjardins!plg.ulaval.ca).
†Present address : Department of Chemistry & Biochemistry,
New Mexico State University, Las Cruces, NM 88003, USA.
to drought stress (Subramanian & Charest, 1995)
and to pathogens (Perrin, 1990). In return, the
mycorrhizal fungi obtain plant carbohydrates for
their growth and maintenance ; c. 10% of carbon (C)
translocated to the roots pass to the fungal partner
(reviewed by Fitter, 1991).
In order to improve plant growth, the benefits
from a mycorrhizal association must be accompanied
by a stimulation of photosynthetic C uptake that will
at least compensate the C lost to the fungus. It is
Printed from the C JO service for personal use only by...
540
D. Louche-Tessandier et al.
generally assumed that enhancement of photosynthetic rates results from increased levels of leaf P
as a result of the mycorrhizal contribution to the P
plant uptake (Fitter, 1991). However, such stimulation of photosynthesis will depend on other
environmental factors such as atmospheric CO and
#
incident light levels. For the host plant, these factors
can therefore modify the balance between the costs,
and the benefits, of a mycorrhizal relationship.
In general, CO enrichment stimulates growth and
#
photosynthesis of C plants (Farrar & Williams,
$
1991). Whereas in the short term, CO enrichment
#
stimulates photosynthesis through the suppression
of photorespiration (Gerbaud & Andre! , 1980), its
long term effects depend on the equilibrium between
production of carbohydrates in source leaves, their
loading into and unloading from the phloem, and
their utilization in sink organs (Farrar & Williams,
1991). In situations of insufficient sink strength
relative to the C assimilation activities, sugars
accumulate in source leaves and trigger a downregulation of photosynthesis that balances the source
and sink activities (Bowes, 1991). It was suggested
that mycorrhizal fungi can represent a significant
sink for any excess of assimilates (Hodge, 1996 ;
Wright et al., 1998) and therefore decrease the plant
susceptibility to down-regulation of photosynthesis
under prolonged exposure to elevated CO . Mycor#
rhizas can further delay down-regulation of photosynthesis by increasing the uptake of nutrients
required to sustain the stimulated plant growth and
the formation of new sink organs (Lewis & Strain,
1996).
There are several examples where CO enrichment
#
increases the percentage of ecto- or vesicular–
arbuscular (VA)-mycorrhizal colonization in tree
and grass species although this effect is somewhat
variable (reviewed by Hodge, 1996 ; see also Rillig
et al., 1998). Stimulation of mycorrhizal colonization
under elevated CO concentrations may be caused by
#
an increase of C allocation to the root system
therefore stimulating root growth, especially the fine
roots which are the main site of mycorrhizal infection
(Hodge, 1996). In in vitro culture systems, enhancement of mycorrhizal colonization may also be
related to the stimulation of hyphal growth by
synergistic effects between high CO concentrations
#
(0n5–1 %) and flavonoid compounds in root exudates
(Chabot et al., 1992a ; Elmeskaoui et al., 1995). In
both cases, an increase in the colonization rate will
increase the total C cost of the mycorrhizal infection
which was assumed to be directly proportional to the
number of infection sites over the whole root system
and to the total mycorrhizal respiration (Fitter,
1991). However in sink-limited plants growing under
high CO , the relative cost of the C exported to the
#
mycorrhizal fungi may decrease despite enhanced
colonization rates as photosynthesis is able to
replenish its C pool under these conditions (Lewis &
Strain, 1996). Therefore, the impacts of a mycorrhizal colonization on the balance between plant
costs and benefits would be determined largely by
the plant source–sink equilibrium.
In this study, we investigated the interactive
effects of CO concentrations (350 and 10 000 ppm)
#
and PPF (60 and 300 µmol m−# s−") on the
relationship between the vesicular–arbuscular
fungus Glomus intraradices and potato plantlets
(Solanum tuberosum) cultured in an in vitro tripartite
system as described by Elmeskaoui et al. (1995). Our
objectives were to determine, under controlled
conditions, how changes in the mycorrhizal
colonization rate and of the plant source–sink
relationship would alter the balance between the
costs and the benefits for the mycorrhizal potato
plantlets. We used a very high CO concentration
#
(1%) because of its stimulating effect on the
mycorrhizal colonization rate in strawberry plantlets
cultured in an in vitro tripartite system (Elmeskaoui
et al., 1995). Also, the use of very high CO
#
concentrations is common in artificial growth conditions such as in vitro culture system (Gouk et al.,
1979) and proposed life support systems in space
(Reuveni & Bugbee, 1997).
  
Plant material
Potato plantlets (Solanum tuberosum L. cv. Lp
89–221 from the experimental farm of the Agriculture and Agri-food Canada at La Pocatie' re, QC,
Canada) were multiplied in vitro and subcultured on
MS solid medium (Murashige & Skoog, 1962) (0n55
mM myo-inositol, 27 µM glycine, 2n4 µM
pyridoxine-HCl, 4n1 µM nicotinic acid, 1n2 µM
thiamine-HCl and no growth regulators). Plantlets
were kept in a growth chamber at a constant
temperature of 23mC and illuminated for 16 h d−"
under a PPF of 60 µmol m−# s−". One wk before
mycorrhizal infection, nodal sections of potato
vitroplants (apex and the four upper leaves) were
excised and transferred on Sorbarod2 cellulose
support (Baum Gartner Paper, Lausanne,
Switzerland) imbibed with 3n5 ml of half-strength
MS liquid medium containing 3% sucrose to induce
rooting.
Fungal inoculum
The fungal inoculum used for the VA mycorrhiza in
this study was G. intraradices Schenk & Smith
(DAOM 197198, Biosystematic Research Center,
Ottawa, Canada). Spores of G. intraradices were
obtained from their co-culture with isolated tomato
roots on minimal White medium according to
Chabot et al. (1992a). For primary colonization (i.e.
the production of G. intraradices inocula in root-
Printed from the C JO service for personal use only by...
Growth and photosynthesis of mycorrhizal potato plantlets
organ cultures), new tomato roots (7 cm long) were
placed in the presence of 200 viable fungal spores
and incubated in darkness at 25mC for 10 wk in 15 cm
polycarbonate petri dishes sealed with Parafilm.
Since tomato root-organ cultures show a lower
colonization rate than carrot root-organ cultures,
200 viable spores rather than 40 as in Elmeskaoui et
al. (1995) were used in order to reduce the time
required for the primary root colonization by the
fungus. Then five healthy lateral roots with intact
root apices were cut into 4-cm sections and placed in
a Magenta2 (Magenta Corp., Chicago, IL, USA)
polycarbonate box for 30 d. At the end of this period,
the roots were 100% infected and hyphae were
visible on the surface of the culture medium and the
walls of the growth container. At this stage, the coculture was ready for the establishment of the
secondary mycorrhiza.
541
(350 and 10 000 ppm), and PPFs (60 and 300 µmol
m−# s−"). The experimental unit consisted of one
Magenta box containing four vitroplants.
For the two experimental blocks, eight Magenta
pots, each containing either four control or four
mycorrhizal potato vitroplants, were placed in 60 l
growth chambers in which normal or CO -enriched
#
atmospheres were circulated at a flow rate ensuring a
complete renewal within 30 min. Illumination was
provided by a combination of incandescent and
fluorescent lamps (60 µmol m−# s−") or by high
pressure sodium lamps (300 µmol m−# s−"). We
assumed that the spectral difference between the two
types of lamps was insignificant compared with the
difference in light intensities. The photoperiod was
16 : 8 hr (light : dark) and the temperatures were
23p1mC (day) and 19p1mC (night).
Biomass determination
Tripartite culture system
The protocol followed for the tripartite culture
system is described in detail in Elmeskaoui et al.
(1995). Briefly, the MS medium was removed by
suction from the Sorbarod plugs supporting the
vitroplants, and thoroughly rinsed with distilled
water and replaced by the minimal M medium
(MgSO :7H O, 2n97 mM ; Ca(NO ) :4H O, 1n22
%
#
$#
#
mM ; KNO , 0n79 mM ; KCl, 0n87 mM ; KH PO ,
$
# %
35 µM ; NaFe-EDTA, 21n8 µM ; MnCl , 30n3 µM ;
#
ZnSO :7H 0, 9n2 µM ; H BO , 24n2 µM ; KI, 4n5 µM ;
%
#
$ $
CuSO :5H O, 0n52 µM ; Na MoO :2H O, 0n01 µM ;
%
#
#
%
#
myo-inositol, 277n5 µM ; glycine, 40n0 µM ; nicotinic
acid, 4n1 µM ; thiamine-HCl, 0n30 µM ; pyridoxineHCl, 0n47 µM ; sucrose, 1% ; Gel-gro, 0n25% (pH
5n5)). This procedure was used to remove the
remaining sucrose in the paper plugs which might
interfere with the establishment of the secondary
mycorrhiza. The secondary colonization (i.e. the
colonization of vitroplants with G. intraradices
already formed on root-organ cultures) was then
established under aseptic conditions by placing four
vitroplants per Magenta container (type GA7) in
contact with sterile (control) or colonized (treatment)
tomato roots. The tripartite culture was conducted
over 4 wk and 10 ml of fresh liquid medium M was
supplied aseptically to each Magenta container
weekly.
Experimental design
A factorial 2i2i2 experiment, with two
randomized complete blocks independent in time,
was designed to study the interactive effects of
environmental factors (CO and PPF) and endo#
mycorrhizal colonization by G. intraradices on
growth and photosynthesis of in vitro potato
plantlets. The factors were ; the in vitro mycorrhiza
(control and G. intraradices), CO concentrations
#
After 14 and 28 d of triculture, one potato vitroplant
per pot was randomly taken for fresh and dry
biomass determination. The plantlets were harvested
with their roots which were carefully removed under
water from their cellulose supports. Fresh and dry
weights of the shoots and roots were measured on an
analytical balance, and used to determine the plant
water content and the dry root : shoot ratio.
Photosynthetic O evolution
#
Light saturation curves of CO -supported O evol#
#
ution were made during the last week of the
experiment on leaf discs from the second and\or
third leaves of potato vitroplants grown under the
different
environmental
conditions
already
described. Photosynthetic O
evolution was
#
measured at 23mC with a LD2 leaf-disc electrode
system (Hansatech Instruments Ltd, King’s Lynn,
Norfolk, UK) in the presence of saturating CO
#
concentrations (5 %) as described by Walker (1989).
After a dark period of 20 min, leaves were
illuminated by an array of red light emitting diodes
(LH36 209 Ultrabright) at a PPF of 240 µmol m−# s−"
until complete induction of photosynthesis
(measured as a stable O evolution rate) was
#
achieved. Then, rates of O evolution were measured
#
after 5 min illumination at each PPF, increasing
from 20 to 1000 µmol m−# s−". The maximum
quantum yield of O evolution (Φmax) was estimated
#
from the linear portion of the light saturation curves
(0–100 µmol m−# s−") and the maximum capacity of
photosynthesis for O evolution (Pmax) was measured
#
at saturating light and CO levels.
#
Chlorophyll-a fluorescence
Simultaneously with O evolution, Chla fluorescence
#
was measured from leaf discs enclosed in the LD2
Printed from the C JO service for personal use only by...
542
D. Louche-Tessandier et al.
leaf-disc electrode system with a PAM 101\103
chlorophyll
fluorometer
(Walz,
Effeltrich,
Germany). After at least 20 min of dark adaptation,
the minimal level of Chla fluorescence (Fo) was
measured with the non-actinic 1n6 kHz modulated
light and the maximal Chla fluorescence level (Fm)
was induced by a 1 sec saturating flash provided by
a KL1500 Schott light source (Schott, Mainz,
Germany). During the saturating flash, the frequency of the modulated measuring light was 100
kHz. After 5 min of illumination at each PPF, the
steady state (Fs) and the maximal (Fmh) fluorescence
levels were measured in a similar way to Fo and Fm.
Immediately after the saturating flash, the actinic
light was turned off and the minimal fluorescence
level (Foh) was measured in presence of a far red light
using a RG715 long-pass filter. From the different
fluorescence levels, the maximum (Fv\Fm) (Adams
et al., 1990) and the operational (∆F\Fmh) (Genty et
al., 1989) quantum yield of PSII electron transport
were determined and the coefficients of photochemical (qP) and non-photochemical quenching
(qN) of chlorophyll-a fluorescence were calculated
according to van Kooten & Snel (1990). The qP
coefficient is an indication of the fraction of PSII
reaction centres which are in an open state, where the
oxidized primary quinone electron acceptor (QA) can
accept, via a pheophytin molecule, an electron
provided from the special Chla molecule P
')!
(Krause & Weis, 1991). Complementary information
is given by the qN coefficient, which indicates the
fraction of absorbed light energy that is dissipated as
heat in competition to the photochemical reactions
and Chla fluorescence emission.
Leaf-pigment contents and stomatal conductance
The Chla and b contents as well as total carotenoids
were measured on the second and third leaves, as for
the O and fluorescence measurements. Leaves were
#
ground in aqueous acetone (80%) and the pigment
concentrations were estimated from absorbance
measurements of the extracts made at 470, 647 and
663 nm, according to Lichtenthaler & Wellburn
(1983). Stomatal conductance was measured on Day
28 of the experiments on the abaxial side of the leaves
with a diffusion porometer (AP4, Delta-T Devices,
Cambridge, UK).
Assessment of arbuscular-mycorrhizal colonization
The roots of plants used for photosynthetic measurements were collected after 28 d of triculture and
stored until staining in a 13 :5 :200 (v\v\v) solution
of formaldehyde (37%) : glacial acetic (99n8%) :
ethanol (50%). Before staining, roots were bleached
in a boiling solution of 10% KOH for 10 min, rinsed
with distilled water, neutralized with 1% HCl and
then rinsed again with distilled water. Roots were
stained in a lactoglycerol solution (50% lactic acid :
water : glycerol, 2 : 1 : 1 w\w\w) containing tryphan
blue 0n05% and heated until boiling. They were
thereafter stored in a lactoglycerol solution (lactic
acid (87%), glycerol (6%) and water (6%)). For root
colonization assessment, countings were made according to the grid-line intersect method (Giovanetti
& Mosse, 1980), using ai40 (model BHZ-RFL-T3,
Olympus Optical Inc., Japan) magnification stereo
microscope. Indistinct blue spots on roots were
screened ati100 magnification.
Statistical analysis
Analysis of variance (ANOVA) were performed on
the variables using the SuperANOVA statistical
program (Abacus Concepts Inc., Berkeley, CA,
USA) according to the General Linear Model.
Before ANOVA, the homogeneity of the variance
was checked using the Levine’s test and the residual
graphic analysis. When required, the means were
transformed to log(xj1) (Steel & Torrie, 1980).

Percentage of mycorrhizal colonization
In potato vitroplants grown in presence of the fungal
inoculum in the tripartite culture system, the
percentage values of root segments showing the
presence of the VA-mycorrhizal G. intraradices
(hyphae, arbuscules or vesicles) after 28 d of
triculture varied from 4n4p0n6% to 7n8p1n0%
depending on the growth conditions (Table 1).
ANOVA of the means indicated that CO -enrich#
ment significantly stimulated the colonization rate (P
l 0n006).
Growth characteristics
The dry matter (DM) of potato plantlets grown for
14 and 28 d under the different experimental
conditions are presented in Table 1. After 14 d of
triculture, there was a significant interaction
(Pl0n001) between the three factors CO , PPF and
#
mycorrhiza. In all treatments, DM production
strongly increased with increasing PPF from 60 to 30
µmol m−# s−". CO -enrichment also stimulated DM,
#
except in mycorrhizal plantlets grown at low PPF
where no difference was observed. However, the
effect of VA mycorrhiza on DM production in potato
vitroplants was highly dependent on the CO and
#
PPF levels during the triculture. Under normal CO
#
concentration, the mycorrhizal fungus did not affect
DM production. However, under a CO -enriched
#
atmosphere, mycorrhizal effects on DM production
depended on the incident PPF : a decrease (k21%)
and a stimulation (j25%) of DM production after 2
Printed from the C JO service for personal use only by...
Growth and photosynthesis of mycorrhizal potato plantlets
543
Table 1. Effects of inoculation with Glomus intraradices and of different levels of CO and photosynthetic
#
photon flux (PPF) on the percentage of roots showing the presence of mycorrhiza, on the production of dry matter
(DM ) and on the root to shoot (R : S) ratio of potato plantlets cultured in an in vitro tripartite system
CO
#
(ppm)
350
PPF
(µmol m−# s−")
60
300
10 000
60
300
Significance (P values)
Factors
CO
#
PPF
Mycorrhiza
CO iPPF
#
CO iMycorrhiza
#
PPFiMycorrhiza
CO iPPFiMycorrhiza
#
Error df
Mycorrhiza
28 d
NM
M
NM
M
NM
M
NM
M
% of
infection
0
4n4p0n6
0
4n8p0n6
0
5n7p0n7
0
7n8p1n0
0n006
0n079
0n0001
0n259
0n006
0n079
0n259
39
DM (mg)
14 d
22n1p4n1
24n0p7n5
58n7p3n3
51n4p8n4
31n6p11
25n0p8n1
90n5p23
113p15
0n001
0n001
0n334
0n001
0n066
0n077
0n001
52
DM (mg)
28 d
42n9p6n1
43n9p9n8
91n8p36
90n6p18
61n4p9n4
43n9p14
122p19
136p36
0n0003
0n0001
0n830
0n020
0n884
0n261
0n169
40
R : S ratio
0n10p0n01
0n11p0n01
0n44p0n07
0n33p0n03
0n10p0n01
0n13p0n02
0n62p0n11
0n84p0n14
0n005
0n0001
0n493
0n011
0n245
0n496
0n390
16
MeanspSE. M, mycorrhizal ; NM, non-mycorrhizal.
wk of growth under low and high PPF, respectively,
were observed in mycorrhizal plantlets compared
with control plantlets.
The stimulatory effects of CO enrichment and
#
increased PPF on DM production (P 0n001) were
also observed after 28 d (Table 1). Whereas the
CO iPPFimycorrhiza interaction observed at 14
#
d was no longer significant (Pl0n169) at 28 d
(despite a 29% decrease and 11% increase in DM
related to mycorrhiza at low and high PPF, respectively, under elevated CO ), there was still a
#
significant interaction between CO and PPF
#
(Pl0n020). The lack of significance of the
CO iPPFimycorrhiza interaction at 28 d could
#
indicate a possible restriction of plant growth
towards the end of the experiment because of
insufficient space in the Magenta containers. After
pooling plants grown in the presence and absence of
the fungal inoculum, examination of the mean values
of plant DM indicates that in the second half of the
experiment (14–28 d of culture), the production of
plant DM at low PPF was 88% of the DM at 14 d for
both normal and high CO concentrations. However
#
at high PPF, this relative increase of DM was 66%
under normal CO and only 26% under high CO
#
#
concentrations. Clearly, the stimulatory effects of
high CO and PPF on growth of potato plantlets
#
could not be sustained during the second part of the
experiment.
In addition to their effects on plant DM, the
growth conditions strongly altered C allocation as
seen by the changes of the root : shoot (R : S) ratio in
potato plantlets (Table 1). ANOVA indicated a
significant interaction of CO and PPF on the R : S
#
ratio (Pl0n0001). Increases of this ratio as PPF
increased (from 60 to 300 µmol m−# s−") were
observed at both CO concentrations whereas the
#
stimulating effect of elevated CO on the R : S ratio
#
was observed only at high PPF and not at low PPF.
The R : S ratio was not significantly modified by the
presence of mycorrhiza (Pl0n453).
Water content and stomatal conductance
The mean values of the water content and stomatal
conductance of the potato plantlets after 28 d of
growth under the different experimental conditions
are presented in Table 2. The PPF was the main
factor affecting plant-water content ; this was consistently lower under high PPF than low PPF.
However the magnitude of the PPF effect was
modulated by the CO concentration and the
#
presence of mycorrhiza, as indicated by the significant triple interaction (Pl0n0003) between the three
factors. The lowest water-content value was found in
the mycorrhizal plants grown under high PPF and
CO .
#
A significant interaction (Pl0n009) between the
three factors on stomatal conductance measured on
day 28 was observed (Table 2). The major factor
appeared to be the mycorrhiza ; the mycorrhizal
plantlets had higher stomatal conductances than
control plantlets, except in those grown under high
PPF and CO . In these plants, the low stomatal
#
conductance indicates that the stomata were mostly
closed at the time of the measurement.
Printed from the C JO service for personal use only by...
544
D. Louche-Tessandier et al.
Table 2. Effects of inoculation with Glomus intraradices and of different levels of CO and photosynthetic
#
photon flux (PPF ) on the plant-water content and on stomatal conductance of potato plantlets cultured for 28 d
in an in vitro tripartite system
CO
#
(ppm)
PPF
(µmol m−# s−")
350
60
300
10 000
60
300
Mycorrhiza
Plant water content
(%)
Stomatal conductance
(mm s−")
NM
M
NM
M
NM
M
NM
M
92n6p0n5
92n8p0n5
84n2p0n9
87n1p0n8
91n4p1n1
93n7p0n6
84n5p1n1
80n5p1n4
23n9p2n5
27n7p2n1
22n6p3n5
30n6p1n0
25n4p1n3
35n1p1n3
2n7p0n4
2n1p0n2
0n006
0n0001
0n219
0n015
0n041
0n420
0n0003
40
0n0001
0n0001
0n0012
0n0001
0n363
0n276
0n009
35
Significance (P values)
Factors
CO
#
PPF
Mycorrhiza
CO iPPF
#
CO iMycorrhiza
#
PPFiMycorrhiza
CO iPPFiMycorrhiza
#
Error df
MeanspSE. M, mycorrhizal ; NM, non-mycorrhizal.
Table 3. Effects of inoculation with Glomus intraradices and of different levels of CO and photosynthetic
#
photon flux (PPF ) on the contents of Chl a, Chl b, Chlajb, and total carotenoids measured in leaves of potato
plantlets after 28 d of culture in an in vitro tripartite system
CO
#
(ppm)
350
PPF
( µmol
m−# s−")
60
300
10 000
60
300
Significance (P values)
Factors
CO
#
PPF
Mycorrhiza
CO iPPF
#
CO iMycorrhiza
#
PPFiMycorrhiza
CO iPPFiMycorrhiza
#
Error df
Mycorrhiza
NM
M
NM
M
NM
M
NM
M
Chl a
(mg g−" f. wt)
Chl b
(mg g−" f. wt)
Chlajb
(mg g−" f. wt)
Carotenoids
(mg g−" f. wt)
2n45p0n25
2n41p0n30
1n38p0n17
1n46p0n08
2n26p0n27
2n67p0n28
0n32p0n06
0n55p0n18
0n85p0n10
0n92p0n11
0n50p0n05
0n48p0n03
0n92p0n10
1n02p0n11
0n15p0n03
0n15p0n03
3n26p0n82
3n32p0n99
1n88p0n56
1n94p0n27
3n18p0n89
3n69p0n95
0n47p0n23
0n84p0n63
0n54p0n06
0n59p0n07
0n36p0n04
0n37p0n03
0n56p0n06
0n64p0n06
0n18p0n02
0n26p0n07
0n009
0n0001
0n436
0n005
0n212
0n701
0n855
39
Pigment contents and photosynthetic activities
Pigment contents in the potato vitroplants grown
under the different experimental conditions are
shown in Table 3. There were marked decreases of
Chla, Chlb and total carotenoid contents in plantlets
grown under high PPF compared with those grown
at low PPF. These decreases were more pronounced
in plantlets grown under both high PPF and CO , as
#
0n003
0n0001
0n098
0n0001
0n214
0n733
0n300
39
0n0001
0n0001
0n122
0n0001
0n237
0n718
0n861
39
0n031
0n0001
0n082
0n0032
0n441
0n986
0n678
39
indicated by the significant interaction PPFiCO
#
between these two factors (P0n005). In leaves
developed under high PPF and CO , leaf chlorosis
#
and in some cases leaf necrosis started to appear
towards the end of the experiments. Although the
PPF and CO treatments did not interact signifi#
cantly with the mycorrhiza treatment, it is noteworthy that leaves from mycorrhizal plantlets grown
under high PPF and CO maintained higher Chlajb
#
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Growth and photosynthesis of mycorrhizal potato plantlets
25
(a)
(b)
(c)
(d)
545
20
Rates of O2 evolution (µmole O2 m–2 s–1)
15
10
5
0
25
20
15
10
5
0
0
200
400
600
800
1000
0
200
400
600
800
1000
PPF (µmol m–2 s–1)
Fig. 1. Light saturation curves of oxygen evolution from leaves of potato plantlets cultured in the absence
(open circles) or presence (closed circles) of the mycorrhizal fungus Glomus intraradices under a photosynthetic
photon flux (PPF) of 60 (a, b) or 300 (c, d) µmol m−# s−" and under CO concentrations of 350 (a, c) or 10 000
#
(b, d) ppm.
and total carotenoid contents (78% and 44%,
respectively) compared with those of nonmycorrhizal plantlets grown under the same conditions.
Light saturation curves of photosynthetic O
#
evolution were measured at saturating CO con#
centration on the upper leaves of potato vitroplants
grown under the different conditions (Fig. 1). For
each curve, we determined the initial slope as an
estimation of the maximum quantum efficiency of O
#
evolution (Φmax) and also the light-saturated rate of
O evolution (Pmax) which represents the maximum
#
photosynthetic capacity (Walker, 1989). The results
for the different treatments are shown in Table 4. As
observed for the pigment contents, there are significant interactive effects of PPF and CO (P0n033)
#
and on the Pmax and the Φmax parameters. In leaves
developed under normal CO concentration, Pmax
#
markedly increased with increasing PPF. The opposite effect was found in leaves grown under high
CO , where an increase of PPF lead to a large
#
decrease of Pmax. No differences between the Pmax
values could be detected between control and
mycorrhizal plantlets.
Similar values for the maximum photochemical
yield of O evolution (Φmax) were obtained for the
#
different treatments, except for both control and
mycorrhizal plantlets grown under high CO and
#
PPF where significant decreases were observed
(Table 4). These results are supported by the Chla
fluorescence Fv\Fm ratio measured after dark
adaptation of the leaves. Here again, leaves developed under high CO and PPF had lower Fv\Fm
#
ratios than leaves from other treatments. The Chla
fluorescence Fv\Fm ratio represent a good estimation of the maximum photochemical yield of PS
II, which has been shown to be closely correlated to
the maximum quantum efficiency of photosynthesis
measured under limiting PPF (Adams et al., 1990).
As observed with the photosynthetic pigments, the
negative effect of high PPF and CO during growth
#
on Fv\Fm was lower in leaves from mycorrhizal
plantlets than those from non-mycorrhizal plantlets
grown under the same conditions.
Further information on the effects of mycorrhizas
and environmental conditions on the photosynthetic
efficiencies of potato plantlets were obtained by the
Chla fluorescence quenching analysis (Schreiber et
al., 1995). In potato plantlets cultivated under
normal CO , increase of PPF during the measure#
ments caused a larger decrease of the effective
photochemical yield of PSII electron transport
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D. Louche-Tessandier et al.
546
Table 4. Effects of inoculation with Glomus intraradices and of different levels of CO and photosynthetic
#
photon flux (PPF ) on the maximum quantum efficiency of O evolution (Φmax), on the maximum quantum
#
efficiency of photosystem II photochemistry (Fv\Fm) and on the maximum capacity of O evolution (Pmax)
#
measured in leaves of potato plantlets after 21–28 d of culture in an in vitro tripartite system
CO
#
(ppm)
350
PPF
( µmol m−# s−")
Mycorrhiza
60
NM
M
NM
N
NM
M
NM
M
300
10 000
60
300
φ
max
( µmol O µmol−" photons)
#
0n073p0n008
0n073p0n003
0n073p0n003
0n066p0n001
0n063p0n006
0n068p0n007
0n037p0n003
0n044p0n014
Significance (P values)
Factors
CO
#
PPF
Mycorrhiza
CO iPPF
#
CO iMycorrhiza
#
PPFiMycorrhiza
CO iPPFiMycorrhiza
#
Error df
Pmax
( µmol O m−# s−")
#
Fv\Fm
0n758p0n005
0n767p0n003
0n725p0n005
0n750p0n014
0n763p0n008
0n761p0n002
0n514p0n054
0n579p0n111
Φ
Fv\Fm
0n007
0n001
0n459
0n007
0n805
0n520
0n667
16
max
0n002
0n009
0n770
0n033
0n386
0n713
0n557
16
15n8p1n2
14n4p0n5
25n0p2n3
24n8p0n9
13n4p0n5
14n6p1n5
9n8p1n6
9n8p0n6
Pmax
0n0001
0n0418
0n936
0n0001
0n592
0n991
0n663
16
MeanpSE.
M, mycorrhizal ; NM, non-mycorrhizal.
0·8
(a)
(b)
(c)
(d)
Quantum yield of PSII photochemistry (∆F/FM′)
0·6
0·4
0·2
0·0
0·8
0·6
0·4
0·2
0·0
0
200
400
600
800
1000
0
200
400
600
800
1000
PPF (µmol m–2 s–1)
Fig. 2. Quantum yield of photosystem II electron transport (∆F\FMh) measured at different photosynthetic
photon fluxes (PPF) in leaves of potato plantlets cultured in the absence (open circles) or presence (closed
circles) of the mycorrhizal fungus Glomus intraradices under a PPF of 60 (a, b) or 300 (c, d) µmol m−# s−" and
under CO concentrations of 350 (a, c) or 10 000 (b, d) ppm.
#
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Growth and photosynthesis of mycorrhizal potato plantlets
1·0
(a)
(b)
(c)
(d)
547
0·8
Quenching coefficients (qp and qN)
0·6
0·4
0·2
0·0
1·0
0·8
0·6
0·4
0·2
0·0
0
200
400
600
800
1000
0
200
400
600
800
1000
PPF (µmol m–2 s–1)
Fig. 3. Values of the photochemical qP (triangles) and non-photochemical qN (squares) quenching
coefficients measured at different photosynthetic photon fluxes (PPF) in leaves of potato plantlets cultured in
the absence (open symbols) or presence (closed symbols) of the mycorrhizal fungus Glomus intraradices under
a PPF of 60 (a, b) or 300 (c, d) µmol m−# s−" and under CO concentrations of 350 (a, c) or 10 000 (b, d) ppm.
#
estimated by the fluorescence parameter ∆F\Fmh
ratio (Genty et al., 1989) in the plantlets grown
under low PPF compared with those from high PPF
(Figs. 2a,c). These more pronounced decreases of
∆F\Fmh in plantlets grown under low PPF and CO
#
were associated with larger decreases of the photochemical qP and larger increases of the nonphotochemical qN quenching coefficients as PPF
increased (Fig. 3a,c).
As observed for the light saturation curves of O
#
evolution, CO -enrichment had no effect on
#
fluorescence parameters in plantlets grown at low
PPF (Figs. 2a,b ; Fig. 3a,b). However, CO -en#
richment had a negative effect on fluorescence
parameters in plantlets grown under high PPF.
When subjected to increasing PPF, ∆F\Fmh and qP
measured from those leaves decreased the most
rapidly and qN increased to the maximum value
(Figs 2d,3d). These results clearly indicate a limitation of photosynthetic electron transport in leaves
developed under these conditions. It is important to
note that these negative effects of high PPF and CO
#
growth levels on ∆F\Fmh and qP were less in
mycorrhizal plantlets than in the controls. The
differences are larger at PPFs near those during
growth (i.e. 300 µmol m−# s−"). Such effects of
mycorrhiza on ∆F\Fmh and qP at high PPF and CO ,
#
although limited, are consistent with their effects on
Chlajb and carotenoid contents, Φmax and Fv\Fm
(Tables 3, 4).
        
In this study, we used the in vitro tripartite culture
system (Elmeskaoui et al., 1995) as a simple model to
determine the importance of two environmental
factors, CO and PPF, on the mycorrhizal association
#
between G. intraradices and potato vitroplants via
their effects on the plant source–sink relationship.
Our results demonstrated that CO enrichment
#
(from 350 to 10 000 ppm) during the tripartite culture
resulted in an increased percentage of mycorrhizal
infection in roots of potato vitroplants. These results
support those of Elmeskaoui et al. (1995) who
observed higher in vitro infection rates of strawberry
plantlets by G. intraradices under elevated CO
#
(5000 ppm) compared with normal CO . The very
#
high CO concentration used in our experiments is
#
consistent with other artificial growth systems. It
was also used in an in vitro culture system where it
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548
D. Louche-Tessandier et al.
stimulated growth of an epiphytic Crassulacean acid
metabolism (CAM) orchid (Gouk et al., 1997). In a
proposed life support system in space, 10 000 ppm
CO had no significant effect on vegetative growth of
#
wheat but decreased seed yield by 37%, probably
because of a partial suppression of respiration
required for grain filling (Reuveni & Bugbee, 1997).
The stimulation of mycorrhizal infection by high
CO concentration during the tripartite culture has
#
two possible origins : CO enrichment stimulates
#
photoautotrophic C uptake and consequently
increases C allocation to the root system and to the
VA symbiont ; high CO concentration acts syner#
gistically with root exudates to stimulate VA-hyphal
growth and thereby increasing inoculum strength
(Chabot et al., 1996 ; Elmeskaoui et al., 1995).
Although we cannot clearly determine the relative
importance of these two possibilities without further
experiments, we suggest that after 4 wk of our
experiments, the latter is the most likely for two
reasons. First, increased PPF under normal CO
#
concentration did not increase the percentage of root
infection despite large increases of maximal photosynthetic capacity, root : shoot ratio and DM production. By contrast, CO enrichment promoted
#
mycorrhizal infection under low PPF with no
significant effects on maximal photosynthetic capacity, root : shoot ratio and DM production. Therefore, it appears that in our experimental conditions,
the stimulation of mycorrhizal colonization by high
CO does not result from an increase of photo#
synthetic C fixation. We must stress that this
conclusion might be different from that reached in
experiments carried out in natural conditions where
the increased CO concentrations generally do not
#
exceed the naturally high CO concentration in the
#
soil atmosphere of c. 1000 ppm (Hodge, 1996). In
these experiments, increased C partitioning to the
root system is considered as the main cause for the
CO -enhancement of mycorrhizal colonization
#
(Hodge, 1996 ; Rillig et al., 1998).
The levels of mycorrhizal infection measured in
potato roots after 28 d of experiments were relatively
low compared with those reported previously for
strawberry plantlets developed in the in vitro tripartite culture system (Herna! ndez Sebastia' , 1998).
Our results, and those from previous studies on
mycorrhizas with potato plants, suggest that potato
is a species showing generally low levels of mycorrhizal colonization but still high morphological and
physiological responses to the presence of the
mycorrhizal fungus. Niemira et al. (1995) observed
yield increases and favourable morphological
changes of prenuclear minitubers of potato developed in the presence of very low levels of mature
mycorrhizal association. Also, it was shown recently
that low levels of mycorrhizal colonization (5–15%)
increased resistance of potato plants to pathogens
such as Fusarium (Niemira et al., 1996).
Our results demonstrate that the CO concen#
tration during the tripartite culture affected not only
the degree of mycorrhizal colonization in in vitro
potato roots but also the impacts of the mycorrhiza
on the growth and physiology of potato vitroplants.
Under normal CO concentration during the tri#
partite culture, no difference could be observed
between control and mycorrhizal plantlets in DM
production, pigment contents and photosynthetic
maximum efficiency and maximum capacity. The
only significant difference we noticed under normal
CO concentration was a higher stomatal conduc#
tance in mycorrhizal plantlets than the controls,
except in plantlets grown under high CO and PPF
#
which showed impaired stomatal function. This
stimulation of stomatal conductance is consistent
with previous results showing an increase of stomatal
conductance and transpiration rate in mycorrhizal
plants (Fitter, 1988). Therefore, the in vitro mycorrhization of potato plantlets under normal CO
#
concentration during the tripartite culture altered
plant water relations but did not affect growth and
photosynthetic characteristics. This implies that
under normal CO concentration, the C cost to the
#
mycorrhizal fungus was balanced with a small
stimulation of in situ photosynthetic rate which
could have been undetected during our photosynthetic measurements made under saturating CO
#
conditions. Recently, Wright et al. (1998) observed a
stimulation of photosynthetic rates in mycorrhizal
clover plants measured at 330 ppm CO without
#
enhancement of plant dry weight, suggesting that the
additional fixed C was allocated to mycorrhizal
fungus.
In contrast to normal CO concentration during
#
the tripartite culture, mycorrhizal colonization under
1% CO had significant effects on the growth and
#
physiology of potato vitroplants. However, these
effects were strongly dependent on the PPF during
growth. Under 1% CO and 60 µmol m−# s−",
#
mycorrhizal plantlets accumulated less DM than
non-inoculated plantlets whereas the opposite was
observed at 1% CO and 300 µmol m−# s−". These
#
opposing results can be reconciled by considering
the plant source–sink status under both growth
conditions. At low PPF, photosynthesis was clearly
source-limited since increase of PPF resulted in
large increases of DM production. In addition,
stimulation under high CO and low PPF of the
#
mycorrhizal colonization rate and a possible increase
of metabolic activity of the mycorrhizal fungi by the
elevated CO concentration (Chabot et al., 1992b)
#
would have increased the total C cost for the infection
and therefore the sink strength (Fitter, 1991 ; Hodge,
1996). Consequently, the mycorrhizal relationship
induced in our experiments under low PPF and high
CO is likely to have caused an imbalance of the
#
source–sink relationship leading to a C deficit for the
host plant.
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Growth and photosynthesis of mycorrhizal potato plantlets
The situation is quite different in mycorrhizal
plantlets grown under both high CO and PPF
#
which showed a slight increase of DM production
compared with non-inoculated plantlets. These high
CO and PPF growth levels lead to typical sink
#
limitation and down regulation of photosynthesis as
can be deduced from the following observations.
$
The relative increases of DM between 14 and 28 d
(j26%) of the experiments under these conditions
were low for both mycorrhizal and control plantlets
whereas the relative increases observed during this
period were much higher for the other growth
conditions (66% and 88%).
$ The highest values of the R : S ratio were measured
in plantlets grown under high CO and PPF.
#
$ By increasing growth PPF, the maximum
efficiency Φmax and maximum capacity Pmax of photosynthetic O evolution as well as the maximum
#
quantum efficiency of PS II estimated by the
chlorophyll fluorescence ratio Fv\Fm all decreased
under CO -enriched atmosphere, which is contrary
#
to normal atmosphere where an increase of growth
PPF stimulated Pmax where Φmax and Fv\Fm remained constant. The large decrease of Fv\Fm is
a valuable indicator of photoinhibitory damage in
these plantlets (Aro et al. 1993).
$ Compared with normal CO concentration, in#
crease of growth PPF under high CO concentration
#
resulted in a more pronounced decrease of photosynthetic pigment contents.
From these different observations, it is clear that the
stimulatory effects of high CO and PPF on growth
#
of potato plantlets could not be sustained for the
whole experiment because of severe sink limitation
which resulted in a down regulation of photosynthesis in both mycorrhizal and non-inoculated
potato plantlets.
A common hypothesis proposed in recent studies
related to the impacts of mycorrhizas on plant
responses to CO -enrichment is that the mycorrhizal
#
partner can significantly increase C sink strength and
consequently decrease host plant susceptibility to
down regulation of photosynthesis associated with
the production of large amounts of carbohydrates in
source leaves (Hodge, 1996). So far no evidence was
found to verify this hypothesis. Lewis & Strain
(1996) observed that the response on Pinus taeda to
CO -enrichment was not modified by the presence of
#
the mycorrhizal symbiont and the P supply. More
recently Lovelock et al. (1997) reported a positive
effect of VA-mycorrhiza on Pmax from leaves of
tropical trees developed under elevated CO . How#
ever, this effect was attributed to an increase of leaf
P content rather than a decrease of sink limitation of
photosynthesis since the sucrose and starch contents
in leaves were significantly increased in the presence
of VA-mycorrhiza. As mentioned already, potato
plantlets developed under high CO and PPF in our
#
549
experiments clearly showed symptoms of down
regulation of photosynthesis. Comparison of growth
and photosynthetic parameters from mycorrhizal
and non-inoculated plantlets grown under these
supra-optimal conditions suggests that mycorrhiza
decreased the severity of the symptoms associated
with sink limitation and down regulation of photosynthesis. In mycorrhizal plantlets compared with
controls we measured higher production of DM,
higher contents of Chlajb, higher contents of
carotenoids, higher maximum efficiency of O evol#
ution (ΦO ) measured at low PPF, higher maximum
#
quantum yield of PS II photochemistry estimated by
the Chla ratio Fv\Fm in dark adapted leaves, and
higher quantum yield of photosynthetic electron
transport ∆F\Fm as well as higher photochemical
quenching coefficient qP when measured at PPF
near the growth PPF (300 µmol m−# s−"). All these
differences, albeit small, are consistent and tend to
demonstrate the potential of mycorrhizal fungi in in
vitro tripartite culture to lessen to some extent the
feedback inhibition of photosynthesis related to sink
limitation in potato plantlets.
The effect of mycorrhiza on sink strength may
have been reduced in our experiments by the
presence of tomato roots in the tripartite culture
system. Indeed, the tomato roots growing on
exogenous sucrose may have contributed to the C
needs of the mycorrhizal fungi, thereby decreasing
their dependence on the photosynthetic C uptake by
potato vitroplants.
The results reported in this study could be relevant
for future experiments aiming to optimize growth
conditions of mycorrhizal potato plantlets in the
tripartite culture. The presence of high levels of both
CO and PPF is detrimental in the long term to plant
#
growth and photosynthetic performances. We
suggest that high CO concentrations should be
#
maintained at least in the first weeks of the triculture
in order to stimulate mycorrhizal colonization and
the PPF adjusted so that the source capacity matches
the sink strength of the plants. From our conclusions,
it could be predicted that the optimum growth PPF
will be higher for mycorrhizal than non-mycorrhizal
plantlets because of the increase of the sink strength
through the presence of the mycorrhizal fungus.
              
This work was supported by an operating grant from
NSERC awarded to Y. D. and made possible by the
Ministry of International Affairs of the Que! bec Government through its scientific and technological cooperation
programme with France.

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