Original article

The effects of ectomycorrhizal statuson carbon dioxide assimilation capacity,

water-use efficiency and response to transplantingin seedlings of Pseudotsuga menziesii (Mirb) Franco

JM Guehl J Garbaye

1 INRA Centre de Recherches de Nancy, Laboratoire de Bioclimatologieet d’Écophysiologie Forestières, 54280 Champenoux;

2 INRA Centre de Recherches de Nancy, Laboratoire de Microbiologie Forestière,F 54280 Champenoux, France

(Received 30 March 1990; accepted 5 December 1990)

Summary — One year-old Douglas fir seedlings, mycorrhizal with Laccaria laccata or with Thele-phora terrestris and grown at two levels of phosphorus in the nutrient solution (10 and 40 mg·l-1 P),were compared for water relations and gas exchange before and after transplanting in non-limitingwater conditions. The results show that i), L laccata is more efficient than T terrestris in increasingphotosynthesis and water use efficiency, ii), phosphorus deficiency reduces photosynthesis and wa-ter use efficiency, iii), the stimulating effect of L laccata on photosynthesis and water use efficiencyis, at least partly, due to the improvement of phosphorus nutrition, iv), the photosynthesis reductionresulting from transplanting is due to a non-stomatal mechanism, and v), the recovery of photosyn-thesis involves the regrowth of the external mycelium of mycorrhizas. These results are discussedfrom the viewpoint of the plant-fungus relationships.

ectomycorrhizae / phosphorus nutrition / CO2 assimilation / water-use efficiency / transplant-ing

Résumé — Effets du statut mycorhizien sur la capacité d’assimilation de CO2, l’efficienced’utilisation de l’eau et la réponse à la transplantation de semis de Pseudotsuga menziesii(Mirb) Franco. Des semis de 1 an de douglas, mycorhizés par Laccaria laccata ou Thelephora ter-restris ont été élevés durant une saison de croissance à 2 niveaux de phosphore dans la solutionnutritive (10 et 40 mg·l-1 P) et ont été comparés du point de vue des relations hydriques et deséchanges gazeux avant et après transplantation (à 2 dates différentes, en octobre et en février) enconditions hydriques non limitantes. A faible niveau de phosphore, les plants inoculés par L laccataavaient une surface foliaire plus importante que les plants mycorhizés par T terrestris (tableau 1) etétaient également caractérisés par des taux d’assimilation de CO2 et d’efficience photosynthétiqued’utilisation de l’eau plus élevés (tableau II et fig 1). La carence en phosphore réduit la photosyn-thèse et l’efficience d’utilisation de l’eau (tableau II, fig 1). L’effet stimulant de L laccata sur l’effi-cience de l’eau est dû, au moins en partie, à l’amélioration de la nutrition en phosphore (fig 7 et 9).

La réduction de la photosynthèse consécutive à la transplantation (fig 2), bien qu’accompagnée parune fermeture stomatique (fig 3), est dûe essentiellement à un mécanisme non stomatique (fig 4) etn’est pas liée à une altération de l’état hydrique et nutritionel (fig 7 et 8) des plants. Le rétablissementde la photosynthèse après transplantation est concomitant à la régénération racinaire (fig 5), mais sondéterminisme implique également la reprise d’activité du champignon (fig 6). Ces résultats sont discu-tés du point de vue des relations plante-champignon.

ectomycorhize / nutrition phosphatée / assimulation de CO2 / efficience de l’eau / transplanta-tion

INTRODUCTION

Ectomycorrhizal symbiosis is essential fornursery-grown conifer seedlings and is de-terminant for plant survival and growth af-ter outplanting (Marx et al, 1977; Le Taconet al, 1988). It is also known that different

fungal associates do not provide the samebenefit in this respect, through mecha-nisms as diverse as improving mineral ab-sorption and assimilation affecting hormo-nal balance in the plant, enhancing thecontact between roots and soil, and pro-tecting roots against disease (Chalot et al,1988). This paper describes and discuss-es the physiological status of one year-oldDouglas fir seedlings, associated with twodifferent ectomycorrhizal fungi and grownat two phosphorous levels, before theywere lifted. The behaviour of the same

seedlings transplanted in controlled condi-tions was also considered.

The results presented here are part of aproject which is aimed at understandingthe role played by the fungal associatesduring the transplanting shock suffered byforest plants when outplanted, even innon-limited water supply conditions (Guehlet al, 1989). Gas exchange parameters(CO2 assimilation rate, transpiration rate,water-use efficiency) were used as physio-logical criteria for monitoring the behaviourof plants with different ectomycorrhizalstatus.

MATERIALS AND METHODS

Plant material

Douglas fir (Pseudotsuga menziesii (Mirb) Fran-co) seedlings were grown in the summer in aglasshouse, in 95 ml containers filled with 1/1 (v/v) vermiculite-sphagnum peat mix inoculatedwith the ectomycorrhizal fungus Laccaria lacca-ta or non-inoculated. Inoculum was myceliumaseptically grown for two months in glass jars, ina vermiculite-peat substrate moistened with nu-trient medium. Twenty per cent (v/v) inoculumwas mixed with the potting mix before filling thecontainers. Each inoculation treatment was wa-tered with a complete nutrient solution contain-ing either 10 or 40 mg·ml-1 phosphorus asNa2PO4. Each fungus-phosphorus level treat-ment involved 120 seedlings. At the end of Sep-tember, when growth stopped and buds wereset up, a random sample of 6 seedlings pertreatment was observed for mycorrhizas with astereomicroscope after gently washing the rootsystems. Ectomycorrhizal development was rat-ed according to a four-level scale (0: no mycor-rhiza; 1: rare mycorrhizas; 2: several conspicu-ous mycorrhizal clusters and/or mycorrhizasdisseminated throughout the root system; 3: my-corrhizas abundant in all parts of the root sys-tem). Three treatments were chosen for subse-quent measurements and analysis:- Tt low phosphorus level, non inoculated, my-corrhizal with contaminant Thelephora terrestris(mycorrhizal rating: 1.6);-TtP: high phosphorus level, non-inoculated,mycorrhizal with T terrestris (rating: 2.4); ]- LI: low phosphorus level, inoculated with Lac-caria laccata, predominantly mycorrhizal with L

laccata (rating: 2.6) and slightly contaminatedwith T terrestris.

Sampling and experimental set-up

The seedlings were kept in a frostless glass-house during winter, without fertilization, underconditions such that aerial growth was stoppedfrom October to March. Two sets of measure-ments were performed: in November and in Feb-ruary. At each date, 20 plants per treatmentwere randomly picked among the 50% tallestones. Before transplanting, 6-8 of these plantswere used for gas exchange measurements andfor determining the phosphorus and nitrogencontent of the needles. The 12 remaining plantswere used for gas exchange measurements andtransplanted as follows: they were immediatelylifted, their roots washed, and mycorrhizal devel-opment was quantified. The growing white roottips were sectioned, and the seedlings wereplanted in sphagnum peat in flat (3 cm thick)containers with a transparent wall allowing ob-servation of the roots. These containers were

placed in a climate chamber under the followingenvironmental conditions: photoperiod, 16 h; airtemperature, 22 ± 0.2°C (d) and 16.0 ± 0.2°C(night); photosynthetic photon flux density (400-700 nm), 400 μmol m-2s-1 provided by fluores-cent tubes; relative air humidity, 60% (day) and90% (night); ambient CO2 concentration (Ca),420 ± 30 μmol·mol-1. They were watered twicea week with the 10 mg·l-1 P nutrient solution inorder to maintain the moisture of the peat nearfield capacity.

Water status, gas exchange, root regenera-tion (number of elongated white tips), and re-growth of mycorrhizal extramatical mycelium(quantified according to the same rating scaleas above) were assessed 4, 11 and 18 d aftertransplanting.

At the end of each experiment, the seedlingswere processed for dry weight and leaf area de-termination. Needles were then oven-dried

(60°C for 48 h) and mineral analyses were per-formed (February only).

Water status and gas exchangemeasurements

Predawn needle water potential (ψwp) was deter-mined on one needle per seedling prior to thegas exchange measurements by means of aScholander pressure bomb specifically devisedfor measurements on individual conifer needles.

For the November experiment, the plantswere taken from the climate room to a laborato-

ry where gas exchange measurements weremade by means of an open system consisting ofthree assimilation chambers connected in paral-lel in which the environmental factors could becontrolled. Measurements were made at 22.0 ±0.5°C air temperature, 10.6 ± 1.0 Pa·kPa-1 leaf-to-air water vapour molar fraction difference,400 μmol·m-2·s-1 photosynthetic photon flux

density (400-700 nm) and 350 ± 5 μmol·mol-1ambient CO2 concentration (Ca).

For the February experiment, gas exchangemeasurements were made in the climate roomwith a portable gas-exchange measurement sys-tem (Li-Cor 6200, Li-Cor, Lincoln, NE, USA).The CO2 concentration in the climate room was

kept constant (Ca = 425 ± 15 μmol·mol-1).Gas exchange parameters (CO2 assimilation

rate, A; leaf conductance for water vapour, g; in-tercellular CO2 concentration, Ci) were calculat-ed with the classical equations (Caemmerer andFarquhar, 1981) taking into account simultane-ous CO2 and H2O diffusion through the stomatalpores. Intercellular CO2 concentration (Ci) calcu-lations were performed in order to assess

whether differences for A between treatmentsand A changes in response to transplantingwere due to chloroplastic or to stomatal factors(Jones, 1985). Previous measurements madeon conifers (unpublished data) did not show anypatch pattern in stomatal closure, so that relia-ble Ci calculations can be performed from leafgas exchange data. More precisely, CO2 assimi-lation rate was considered in an (A, Ci) graph asbeing at the intersection of two functions: i), thephotosynthetic CO2 demand function (D) whichdefines the mesophyll photosynthetic capacityand, ii), the photosynthetic CO2 supply function(Su) defining the diffusional limitation to CO2 as-

similation. For determining the (D) functions, Cawas varied stepwise and A and Ci were calculat-ed for each step. The Su function is a line withan x-axis intercept approximately equal to Caand a negative slope approximately equal to -g(Guehl and Aussenac, 1987). Water-use effi-

ciency (WUE) was determined as the A/g ratio.At the end of the experiment, the seedlings

were harvested and plant material was separat-ed into different compartments (needles, stemsand root systems). Each compartment wasoven-dried at 60°C for 48 h and weighed. Thedried needles were kept for mineral analysis.

Projected needle areas of the seedlingswere determined with a video camera coupledto an image analyser (ΔT area meter; ΔT devic-es, Cambridge, UK).

Mineral analyses

The total nitrogen content of the dried and

ground needles was determined with a C/Nanalyser (Model 1500; Carlo Erba, Italy). Thevalues obtained with this technique are about10% higher than those obtained with the Kjel-dahl method. The phosphorus concentrationswere determined after pressure digestion of theground material with 100% HNO3, at 170°C for6 h (Schramel et al, 1980) with a direct currentplasma emission spectrometer (Model SpectroSpan 6; Beckman Instruments, USA).

RESULTS

Plant size and biomass

Data relative to the size and biomass ofthe February seedlings (before transplant-ing) are given in table I. Stem height washighest in the TtP and LI treatments. Rootcollar diameter and total dry weight weresignificantly higher in TtP than in the othertreatments, whereas there was no signifi-cant difference in the root/shoot ratio be-tween the different treatments. Needlearea was significantly higher in TtP and LIthan in Tt. The seedlings of the differenttreatments did not exhibit significant differ-ences in their specific leaf dry weight (ratioof needle dry weight to needle area).

Gas exchange and water-use efficiency

Table II gives the mean values of CO2 as-

similation rate (A), stomatal conductance(g) and water-use efficiency (WUE = A/g)in the different treatments before trans-

planting, in the 2 experiments. TtP and LIexhibited A values significantly higher than

those in Tt both in November and in Febru-

ary. A was higher in TtP than in LI in No-vember but not in February. In November,TtP was characterized by g values signifi-cantly higher than those in the other treat-ments, while in February there was no sig-nificant difference for this parameter.

Water-use efficiency in TtP and LI wassignificantly higher than that in Tt in both

experiments. There was no significant dif-ferences between TtP and LI. For a giventreatment, the WUE values were identicalfor the two experiments.

Figure 1 gives an insight into the WUEregulation at the individual level prior totransplanting. The regression lines wereforced through the origin so that their

slopes (water-use efficiency) could be

compared. In November as well as in Feb-ruary, the invididual variability of the plotsrelative to treatments TtP and LI was or-dered along the same linear relationshipexpressing proportionality between A andg and thus constancy of WUE both for theindividual plants and the two dates. In con-

trast, treatment Tt did not exhibit such acontrol of WUE at the individual level since

no significant (P < 0.05) correlation be-tween A and g was observed for this treat-

ment. Moreover, the plots of the latter

treatment occupied a lower position in the(A, g) graphs, thus indicating lower WUE.

Transplanting resulted in a marked de-crease of A between day 0 and day 4 in alltreatments and for the 2 measurement pe-riods (fig 2). In February, the decrease of Acontinued until 18 d after transplanting fortreatment LI, while a slight recovery of Awas observed from d 4 in treatments Tt

and TtP. Such a recovery was not appar-ent in November, when the decrease in Awas more pronounced in the TtP seedlingsthan it was in the LI seedlings, since the Avalues of these treatments were signifi-cantly different at day 0, but were not dif-ferent 18 d after transplanting (fig 2). In

February, a very different pattern was ob-served with the decrease of A being themost pronounced in LI.

Transplanting also affected g (fig 3) in amanner approximately identical with the ef-fects on A. However, the decrease of gwas less pronounced than that of A, partic-ularly during the first 4 d after transplant-ing. In February, the recovery of g in treat-ments TtP and Tt took place only from d11, and a recovery of g was also observedin treatment LI.

In figure 4 the gas exchange data of fig-ures 2 and 3 are presented in A vs Cigraphs. For both measurement periodsand in all treatments the decline of A in re-

sponse to transplanting was accompaniedby increasing Ci, and was primarily due to

alterations in the photosynthetic demandfor CO2 while the supply function (relatedto stomatal conductance) was affected

only to a minor extent.

Root and mycorrhizal regeneration

Root (fig 5) and mycorrhizal (fig 6) regener-ation of the transplanted seedlings oc-

curred from d 11 after transplanting in No-vember, and from d 4 in February. Rootregeneration was the highest in treatmentTtP for both periods and was markedlylower in the other treatments (fig 5). The

seedlings of treatment TtP also had the

highest mycorrhizal regeneration in Febru-ary (fig 6), but not in November. Mycorrhi-zal regeneration in the LI treatment wasidentical to that in TtP and superior to thatin Tt in November, but was noticeably low-er than that in the other treatments in Feb-

ruary.

Water and nutrient status

No significant alteration in &psi;wp was ob-served after transplanting in any of thetreatments and all treatments had similar

&psi;wp values ranging from -0.8 to -0.6 MPa(data not shown).

Before transplanting, needle P concen-tration was significantly higher in the TtPseedlings than in the other treatments (fig7). Treatments Tt and LI had identical nee-dle P concentrations in November, while inFebruary the needle P concentration wasslightly but significantly higher in LI than inTt. In February, transplanting significantly

reduced the needle P content in TtP, whilethis concentration remained unchanged inthe other treatments.

Needle N concentration in the LI treat-ment was significantly lower than those oftreatments Tt and TtP in November andlower than in TtP in February (fig 8). Theseedlings of treatment Tt had higher Nconcentrations in February (fig 8). Theseedlings of treatment Tt had higher Nconcentrations in February than in Novem-ber, while no seasonal changes occurredin the other treatments. Transplanting hadno significant effect on needle N concen-tration in any of the treatments.

Gas exchange parameters of the indi-vidual plants were examined with respectto their needle nutrient status. There was

no relationship between these parametersand the needle N concentrations. Therewas a significant correlation between Aand needle P concentration only in treat-ment Tt (fig 9a), in the other treatments Awas not related to P. Stomatal conduc-tance was significantly correlated with Pvia a parabolic function (fig 9b), with theminimum of g occurring at about 2000

&mu;g·g-1 P in the needles. The clearest pic-ture of limiting effect due to P was ob-served relative to the WUE data shown in

figure 9c: there was a close linear relation-ship between WUE in treatment Tt, whilethe plots relative to treatments LI and TtPoccupied the non-limiting P region (P con-centration superior to 700 &mu;g·g-1) of thegeneral relationship.

DISCUSSION

The seedlings associated with T terrestrisand supplied with a non-limiting (40

mg·l-1 (P) nutrient solution were taller andhad a higher biomass that the seedlingsassociated with T terrestris but suppliedwith a 10 mg·l-1 (P) solution. Seedlingsmycorrhizal with L laccata and grown un-

der limiting P conditions (10 mg·l-1 P)were taller than the seedlings infected withT terrestris and supplied with the same so-lution (table I). However, both root collardiameter and total plant biomass were notsignificantly different between the two lat-ter treatments. Harley and Smith (1983)and Guehl et al (1990) have reported simi-lar results indicating i), that the extent towhich growth was affected by ectomycor-rhizal infection will depend on the fungalspecies and strain used as mycobiont andii), that there may be a discrepancy be-tween effects of mycorrhizae on stem el-ongation on the one hand and on diameterand weight growth on the other. Tyminskaet al (1986) observed higher biomass

growth in Pinus silvestris seedlings infect-ed with L laccata than in seedlings infect-ed with T terrestris over a wide range of Pconcentrations in nutrient solution (0.1-31mg·l-1). These authors also observed thatthe difference in biomass between the twotreatments was not accompanied by a sig-nificant difference in needle P concentra-

tion, and suggested the stimulating effectof Laccaria laccata - even observed in

seedlings with a low percentage of mycor-rhizal roots - to be related to the capacityof this fungus to produce growth regulatorssuch as indole acetic acid (IAA). They sup-ported this assumption by the work of Eket al (1983) who found that the samestrain of L laccata produced large quanti-ties of IAA. In the present study with Pseu-dotsuga menziesii as the host plant,significant differences in needle P

concentrations were found between Tt andLI (figs 7 and 9). Furthermore, needle Pconcentration in LI was intermediate be-tween those in Tt and TtP. That the growthstimulating effect of Laccaria laccata is

mediated, at least partially, by a P nutri-tional effect cannot be precluded here.

In the present study, the superiority of Llaccata as compared to T terrestris was

also observed relative to the CO2 assimila-tion characteristics of the seedlings at theend of the first growing season. As com-pared with the Tt seedlings, needle surfacearea (table I) and CO2 assimilation rates

(table II) of the LI seedlings were about 42and 48% higher, respectively, thus confer-ring to the LI seedlings a whole plant CO2assimulation capacity about 2.1 times thatin the Tt seedlings and approximatelyequivalent to that in the TtP seedlings.Several authors (Jones and Hutchinson,1988; Guehl et al, 1990) have reportedsimilar modulations of host plant CO2 as-

similation capacity due to the nature of themycobiont. CO2 assimilation rate was

clearly P limited in treatment Tt (fig 9a).Using 31P nuclear magnetic resonance,

Foyer and Spencer (1986) studied the ef-fects of reduced phosphate supply on in-tracellular orthophosphate (Pi) distributionand photosynthesis in Hordeum vulgareleaves. They observed that i), over a widerange of leaf Pi, the cytoplasmic Pi level ismaintained constant, while the vacuolar Piis allowed to fluctuate in order to buffer thePi in the cytoplasm and ii), that an overallminimum cytoplasmic Pi concentration ofbetween 5-10 mmol·l-1 is required to sus-tain optimal rates of photosynthesis in thelight. Despite the relatively high P concen-trations found in our study in all the LI andTtP seedlings, some seedlings of thesetreatments exhibited very low A values (fig9a). Thus, other limiting factors are likely tobe involved.

Water-use efficiency was higher andless variable in LI than in Tt (table II, fig 1).Guehl et al (1990) observed that Pinus pin-ea seedlings associated with different ec-tomycorrhizal fungi were characterized byhigher and less variable WUE values thannon-mycorrhizal plants. This result is of

great importance, since it indicates that ec-tomycorrhizal infection may confer en-

hanced drought adaptation to the host

plant, not only by improving water uptake(Druddridge et al, 1980) and plant waterrelations (Boyd et al, 1985), but also

through higher WUE. In the present study,the data of figure 9c suggest that the im-provement of WUE in the L laccaria infect-ed seedlings as compared to the T terres-tris seedlings is mediated by a nutritional Peffect involving both effects on A (fig 9a)and g (fig 9b). It is worth noting that therewas a clear tendancy for g to be increasedwhen total leaf P was lower than 2 000

&mu;g·g-1. In Zea mays, Wong et al (1985)observed a dramatic decrease in A without

any effect on WUE (A/g ratio) when P inthe nutrient solution was decreased from41 to 1.2 mg·l-1. However, in Pinus radia-ta, Conroy et al (1988) found lower WUE inP deficient plants (needle P concentration700-800 &mu;g·g-1) than in non deficient

plants (needle P between 1 000 and 1 500

&mu;g·g-1). Thus, their critical value (800ug·g-1) was the same as in our experi-ments. Harris et al (1983) found that in leafdiscs of Spinacia oleracea, low Pi led to aloss of stomatal control and wide stomatal

apertures, while high Pi induced stomatalclosure. In the same species, Herold

(1978) observed that mannose and deoxy-glucose induced wilting by metabolicallysequestering Pi. Feeding Pi deficient Hor-deum vulgare and Spinacia oleracea cutleaves with Pi through the xylem transpira-tion flow, Dietz and Foyer (1986) observeda short-term (5 min) increase in CO2 as-

similation and a concurrent decrease in

transpiration, resulting in a marked in-

crease of WUE.

Transplanting markedly reduced A in alltreatments in both experimental periods(fig 2). Analysing gas exchange data in Avs Ci graphs (fig 4) clearly established thatthis decline of A occurred while the diffu-sional CO2 supply to the chloroplasts wasenhanced (Ci increased), thus indicatingthat the changes in A were primarily due to

alterations of the mesophyll photosyntheticcapacity. Guehl et al (1989) reached thesame conclusions with transplanted Ce-

drus atlantica seedlings. Our results alsoindicate that the decline in A cannot be ac-counted for by alterations in plant waterstatus and in needle nutrient status (N andP). The only significant effect of transplant-ing on needle nutrient status was the de-crease found for P in the TtP seedlings inFebruary, in which the recovery of A after

transplanting was most marked. The na-ture of the factor triggering the decline of Aremains unknown. In a previous study(Guehl et al, 1989) it has been establishedfor transplanted Cedrus atlantica seedlingsthat the recovery of A, following the initial

phase of decline, was concomitant withroot regeneration. The results obtainedhere (figs 2, 5, 6) suggest that the recoveryof A was related to the recovery of mycor-rhizal activity rather than regeneration of

elongating non-mycorrhizal white root tips.Two mechanisms could be involved: pro-duction of growth regulators by the grow-ing fungus, and/or improvement of waterand mineral uptake through the re-

establishment of mycelial connections be-tween the root and the soil. Our resultsalso show that the ability of the plants toregenerate mycorrhizae after transplantingis affected by seasonal parameters as wellas their ability to regenerate roots (Ritchieand Dunlap, 1982).

ACKNOWLEDGMENTS

This work was supported by a grant from the Of-fice National des Forêts. The authors are grate-ful to R Zimmermann from the University of Bay-reuth (FRG) for mineral analyses. They wish tothank JL Churin, B Clerc, JM Desjeunes, PGross and F Willm, INRA Nancy, for their techni-cal assistance and JL Muller for drawing the fig-ures. They are grateful to Pr B Dell (Murdochuniversity, Perth, Australia) for reviewing the

manuscript.

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