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Physiology and metabolism of ectomycorrhizae

C. Bledsoe1

U. SanqwarD. Brown, S. Roge

M. Coleman15 and J. Amn

W. Littke2

ati5P. Rygiewicz3

U. Sangwanit4 S. Rogers J. Ammirati5

1 College of Forest Resources AR-f 0, University of Washington, Seattle, WA 98 t 95 U.S.A.2 Weyerhaeuser Corp., Centralia WA, U.S.A.,3 US EPA, Corvaltis, OR, U.S.A.,4 Forest Biology, Kasetsart University, Bangkok, Thailand, and5 Botany Dept., University of Washington, Seattle, WA, U.S.A.

Introduction

Managedforests are the forests of today.In these forests, growth and yield are

improved by forest fertilization. Applicationof fertilizers, often nitrogen, has created a

need for more understanding of how min-eral nutrients, roots and soils interact. Thisneed has produced new partnershipsamong forest soil scientists, root physiol-ogists, soil microbiologists, tree nutri-tionists

and mycorrhizal research workers.The study of mycorrhizae is a critical inter-face in understanding the processes bywhich nutrients are transferred from thesoil through fungal hyphae into roots, thenmetabolized and distributed throughoutthe tree. This interface between root and

fungus is illustrated in Fig. 1.

The following is a discussion of ectomy-corrhizal fungal physiology and its effectson coniferous trees, particularly effects on

nutrient uptake, tree nutrition and waterstress. This discussion focuses on 10

years of research conducted by our

mycorrhizal group in forestry at the Uni-

versity of Washington. Our research pro-

gram has focused on two central ques-tions: How do ectomycorrhizal fungi affect

processes of nutrient uptake by forest tree

species? And, do fungal species differ in

their abilities to affect physiological pro-cesses in general?

Nutrient uptake and metabolism

Inorganic nitrogen uptake

Inorganic ammonium and nitrate are as-

sumed to be the major forms in which

nitrogen is taken up by tree roots. Forestsoils generally contain more ammoniumthan nitrate, although levels of either ionare relatively low. Although organic nitro-

gen is also an important form of nitrogen,more attention has been directed to inor-

ganic forms. Soil pH is both affected byand affects uptake of ammonium and



nitrate. With ammonium uptake, hydrogenions are released into the rhizosphere,

while uptakeof nitrate results in

hydroxylion release. These exchanges balance

charge in the roots and substantially alter

pH around the roots. This affects availabi-

lity and uptake of many ions (particularlyphosphorus).

In our lab, we measured uptake ofammonium and nitrate by 3 conifer spe-cies (Douglas fir, western hemlock andSitka spruce) which were either non-

mycorrhizal or mycorrhizal with Hebelo-ma crustuliniforme (Bull. ex. Fr.) Que!.(Rygiewicz et al., 1984a, b). Seedlingswere grown in solid media, then transfer-

red to solutions for short uptake periods.Uptake rates decreased with increasing

acidity,so that

ratesat

pH 3were

only50-70% of rates at pH 7. Mycorrhizalplants generally had higher uptake ratesover the entire pH range, particularlyDouglas fir. Mycorrhizal effects were muchmore noticeable for ammonium uptakethan for nitrate uptake. Unlike many cropspecies, uptake rates for ammonium were

higher, about 10-fold, than nitrate uptakerates. Since ammonium levels in forestsoils are gene-rally higher than nitrate,higher uptake rates might be expected.

Mycorrhizal roots did not release as

much H+ per ammonium taken up as did



non-mycorrhizal roots. This finding sug-gested that mycorrhizal fungi may bufferammonium uptake, allowing uptake tocontinue at faster rates

by reducingacidifi-

cation in the rhizosphere. Another interest-

ing observation was that ions were not

only being taken up by roots, but were

also being released - sometimes in sub-stantial amounts. Potassium efflux was

noted. Clearly, loss or efflux of ions mustbe a temporary phenomenon, since plantsincrease in size and nutrient content over

time. Our results simply indicated thatinflux or efflux of a particular ion maychange from time to time, depending uponconditions. We have shown cation efflux

during a period of rapid ammonium uptakeby Douglas fir roots (Cole and Bledsoe,1976). When all the ammonium in thesolution was depleted, cations were reab-sorbed. Although it may seem inefficient,plant roots both take up and release ionsat rapid rates. Some ions are certainlyretained, but this may be a small percent-age of the total flux.

Potassium fluxes in roots

Our interest in ionic fluxes into and out ofroots led to a study of mycorrhizal effectson these fluxes. Using a compartmentalanalysis technique, we labeled Douglas firroots for 24 h with radioactive rubidium(potassium tracer) (Rygiewicz and Bled-soe, 1984). After labeling, rubidium effluxwas tracked for 10 h. Mathematical anal-yses of efflux data allowed data to be

separated into fluxes and pool sizes for 3

compartments: cell wall/free space, cyto-plasm and vacuole.

There was rapid influx and efflux of

potassium. About 95% of all potassium

enteringroots was

subsequentlyreleased;

net accumulation was only 5% of total flux.

Mycorrhizal fungi did alter fluxes, withmore storage of potassium in the vacuoles

of mycorrhizal root cells. Half-lives of

potassium in all 3 cellular compartmentswere increased by mycorrhizal fungi. For

example,in the vacuolar

compartment,the

half-life was 25 h for mycorrhizal roots, but

only 6.6 h for non-mycorrhizal roots.These data suggest that mycorrhizal fungican alter ion fluxes through roots, reducingefflux and resulting in increased retentionin the roots. Higher fungal metabolic rates

may increase energy for active uptake andretention of ions.

Cation-anion balance

These mycorrhizal effects on ionic fluxesled us to ask whether mycorrhizal fungican change total ionic flux into cells. Acation-anion balance sheet was deter-mined for mycorrhizal and non-mycorrhizalDouglas fir seedling roots during a short

uptake period (Bledsoe and Rygiewicz,1986). Influx and efflux of cations (ammo-nium, potassium, calcium, H+) and anions(phosphate, sulfate, chloride and bicarbo-nate) were measured using stable and

radioisotopes and chemical analyses.In this experiment, mycorrhizae had little

effect on total fluxes, but they did increaseanion uptake and bicarbonate release. Forall treatments, cation fluxes were muchmore rapid than were anion fluxes; 25times more cations enter and leave rootcells than anions. This massive cationinflux was not balanced by parallel anioninflux, but by efflux of H+ and potassium.The very small amount of anion influx was

balanced by bicarbonate efflux. Mostcations were presumably stored invacuoles as salts of organic acids. Ourcalculations suggest that both mycorrhizaland non-mycorrhizal coniferous roots syn-thesize large amounts of organic acids.

Using data from the literature, we com-

pared our data on coniferous roots tothose on several major crop species and



found a major difference. Coniferous rootstake up cations at about twice the rate ofherbaceous crop species -27 vs 14

microequivalents per gram dry wt. of roots

per hour. Since hydrogen ions are the pri-mary ion released to balance cation up-take, coniferous roots acidify the externalmedium (or soil) to a much greater extentthan do roots of crop species. Coniferroots also synthesize greater quantities of

organic acids than do crop species. TableI shows these fluxes.

Organic nitrogen

As indicated earlier, little attention hasbeen paid to organic nitrogen uptake byplants or to fluxes and pool sizes ofsoluble organic nitrogen in forest soils.Early work by Melin in the 1950’s demon-strated amino acid uptake by mycorrhizalroots. There have been few reports since

then.We

investigated uptakeand utiliza-

tion of organic nitrogen, since this path-way may be important for carbon and

nitrogen assimilation by roots.

Amino acid uptake

Using 3 different amino acids, uptakerates by roots of Douglas fir and western

hemlock were measured in solution cul-ture (Sangwanit and Bledsoe, submitted;Bledsoe and Sangwanit, submitted).Seedlings were either non-mycorrhizal or

mycorrhizal with Cenococcum geophilumFr., H. crustuliniforme, or Suillus granula-tus (L.: Fr) Kuntze. Net charge of theseamino acids was either neutral (alanine),plus (aspartic acid) or minus (arginine) atpH 5.5, the uptake solution pH.

Uptake was measured by appearance inroots of [14C)arninoacids. The use of axe-

nic seedlings precluded microbial degra-dation of the amino acids, which wouldhave separated 14C label from the aminoacid. Ionic charge had little effect on rates,since they were similar - about 50 nmol

per mg of root per hour x 10-2 (Bledsoeand Sangwanit, submitted). The singleexception was lower rates (25 nmol) for

arginine uptake by hemlock. Similarly,choice of host species had little effect on

uptake rate, with the exception notedabove for hemlock and arginine. Fungaleffects were significant. Compared to non-

mycorrhizal seedlings, rates for seedlingsmycorrhizal with Hebeloma and Cenococ-cum were 25 and 33% higher, while rates

for Suilluswere

lower - only75%

of thecontrol rates. Thus choice of the fungalpartner did affect amino acid uptake rates.

Amino acid metabolism

Metabolism of these 3 amino acids and a

4th amino acid, glycine, was affected bothby type of amino acid and by mycorrhizae.

After a 4 huptake period,

littleglycine

hadbeen metabolized (90% unaltered gly-cine). In contrast, about 50% of the ala-nine was converted into non-amino carbon



compounds; less than 30% alanine re-

mained. About 70 and 50% of arginineand aspartate, respectively, remained.When root extracts were chromatograph-ed on thin-layer chromatograms, many1aC_labeled compounds were found.Mycorrhizal roots often contained 14C-la-bel not found in non-mycorrhizal roots -such as ’C7’ in Fig. 2. This unknown com-

pound was produced in 5 of the 6 mycor-rhizal treatments, but not in non-mycorrhi-zal (NM)ones. These mycorrhizal-specificcompoundswere not identified.

Amino acid transfer and storage

Mycorrhizal roots might be expected tostore amino acids in fungal tissues and totransfer less to the stele, in contrast to

non-mycorrhizal roots. Using [14C]glycine(Sangwanit and Bledsoe, submitted), we

found that non-mycorrhizal roots did trans-

fer much of their glycine directly to theshoot, whereas mycorrhizal roots storedmore glycine in the roots.

Using microautoradiography, the loca-tion of glycine in root tissues was deter-mined (Sangwanit and Bledsoe, submit-ted). In a time series uptake experiment,mycorrhizal and non-mycorrhizal Douglasfir roots were exposed to glycine for 1, 4,12 and 24 h. Then root tips were frozen in

liquid N2, freeze-dried at -70°C, andvacuum-embedded with a low viscosity,non-water soluble resin (to prevent move-

ment of water-soluble glycine). After cut-

ting ultramicrotome sections, root sectionswere covered with a film emulsion andstored. After photographic development,black dots on the film indicated [14C]gly-cine in root tissues.

Glycine appeared in the stele of non-

mycorrhizal roots at 1 h; transport con-

tinued throughout the 24 h experiment(Sangwanit and Bledsoe, submitted). Formycorrhizal roots, however, much of the

glycine was stored in the fungal mantle.Gradually, glycine was transferred to the

stele over the 24 h experiment. Theseresults indicate that mycorrhizal roots can

serve as a storage organ for organic nitro-



gen in roots. Perhaps in a forest soil, or-

ganic nitrogen may be taken up directly bythe fungi and stored in the mantle. At latertimes, these amino acids could be used as

a source of both carbon and nitrogen forfungal growth as well as for root or tree

growth.

Fungal physiological diversity

Our previous discussion has documentedthe beneficial effects of mycorrhizae on

nutrient uptake and metabolism. Theseresults led to the following question. Do

fungal species differ in their abilities toaffect physiological processes in general?For example, there are a large number of

fungal species - more than 1000 - thatmay form mycorrhizae with Douglas fir

(Trappe, personal communication). Whyare there so many different fungi? Do theyhave different ecological niches? Do they

carry out different functions in associationwith tree roots? This puzzling fungal di-

versity is the focus of our current work.

Identification of fungi on roots

Before studying fungal diversity, it is

necessary to know whether diversity offungal fruit bodies is related to mycorrhizal

diversity. Are

fungiwhich form fruit bodies

also functioning mycorrhizae? We are stu-

dying fruiting patterns and mycorrhizal pat-terns on roots in the same field plots(Rogers, personal communication). Ifthere are correlations between fruiting pat-terns and root-associated mycorrhizalfungi, then we can assume that taxonomicdiversityis related to mycorrhizal diversity.In order to know which fungi are presenton roots, it is necessary to identify root

fungi. However, fungal taxonomy is basedon characteristics of the fruit body. It is

very difficult to characterize root-associ-ated fungi based solely on color and cul-ture characteristics (Bledsoe, 1987) andmany mycorrhizal fungi have not been

grown in culture.We are developing an identification pro-

cedure based on rDNA patterns (Rogerset al., 1988). Using about 1-100 mg fromfruit bodies (fresh or dried), fresh-culturedfungal mycelia or mycorrhizal roots, rDNAwas extracted using a CTAB microprepa-ration method (Fig. 3). After extraction and

purification, the DNA was restricted withEcoRl, run on an agarose gel and South-

ern blots were made with a yeast pBD4probe.

Fig. 4 shows an autoradiograph of rDNA

blot-hybridizatio!n patterns from mycorrhi-zae of Rhizopogron vinicolorA.H. Sm: lane1 = mycelial culture only; lane 2 = Douglasfir mycorrhizal roots; lane 3 = uninfectedroots. The position of fungal bands was

separate from those of the conifer roots.Thus, the fungus infecting the root could

be identified by comparison to patternsfrom a ’library’ of mycorrhizal fungal pat-terns. Since fungal and root patterns didnot overlap, it is not necessary to separatefungal and root tissues before DNA extrac-tion - a considerable advantage.

Fungal physiological diversity

Although many fungi fruit in associationwith Douglas fir, little is known about which

fungus is appropriate for any set of envi-ronmental conditions. We studied one

aspect of physiological diversity - the abil-

ity of fungi to tol,erate water stress (Cole-man et aL, 1988). Over 50 isolates were

tested in pure culture, using polyethyleneglycol to adjust medium water potential.

In response to stress, 3 different growthpatterns were observed (Fig. 5). For typeI, fungi were intolerant of stress and grew



only at the lowest level of stress (-0.02MPa). For type II, fungi did tolerate some

stress. Growth rates decreased with

increasing stress; maximum growth occur-

red in the lowest stress level. For type III,

fungiwere much more tolerant of stress

and even grew faster at a moderate stresslevel. Laccaria spp. were type I. Most ofthe isolates (80+%) were type II. Only 7isolates were type III, including C. geophi-lum and H. crustuliniforme (Coleman et

al., 1988). These results indicate that fungido differ in their abilities to grow under

imposed water stress in pure culture. We

have synthesized mycorrhizal seedlingswith some of these isolates and are stu-

dying their effects on the water relations ofDouglas fir seedlings (Coleman, personalcommunication).



Summary and Conclusions

In addition to our work, other papers pre-sented at this symposium report on

mycorrhizal physiology. For example, thesoils work in Germany by Ritter andcoworkers shows effects of liming soils on

species diversity of fungi. In nutritional stu-dies, Rousseau and Reid from Florida,USA, have focused on phosphorus uptakeand translocation, while Vezina et al., fromLaval, Quebec, evaluated metabolism of

nitrogen supplied in different forms. More

detailed metabolic studies at the Univers-ity of Nancy by Chalot and coworkersshowed more efficient uptake of am-

monium by mycorrhizal plants. Not onlynutrients but also carbon biochemistry isaffected by mycorrhizae as reported byNamysl et al., also at the University of

Nancy. Several papers discussed inter-actions in the rhizosphere, such as El-

Badaqui et al.’s report on mycorrhizal pro-duction of extracellular

phosphatases.Succession of different mycorrhizal typeson seedlings and young trees was report-

ed by Blasius et al. These research resultsillustrate the intense interest in under-standing how mycorrhizae affect hostnutrition and physiology.

With the use of new techniques and

methods, we are now able to understandnot only how fungi affect nutrient uptakeand tree nutrition but also to study more

specific effects of individual fungal spe-cies. In the future, we expect that we willunderstand fungal physiological diversitysufficiently to be ;able to select certain fun-

gal partners for specific field and environ-mental conditions. New areas of research

will probably include: host-fungus recog-nition, genetic engineering of mycorrhizalfungi, studies of :>patial patterns of mycor-rhizal roots in forest soils and microbialinteractions in the rhizosphere.

Acknowledgments

We appreciate the technical assistance provid-ed by Suzanne Bagshaw, Faridah Dahlan, KellyLeslie, Kim Do and HCathyParker.



References

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Bledsoe C. & Rygiewicz P.T. (1986) Ectomycor-rhizae affect ionic balance during ammonium

uptake by Douglas fir roots. New Phytol. 102,271-283

Cole D.W. & Bledsoe C.S. (1976) Nutrientdynamic of Douglas fir. IVI IUFRO WorldCongress Proceedings, Div. II, pp. 53-64

Coleman M.D., Bledsoe C.S. & Lopushinsky W.

(1989) Pure culture response of ectomyc. fungito imposed water stress. Can. J. Bot. 67, 29-39

Littke W.R., Bledsoe C.S. & Edmonds R.L.(1984) Nitrogen uptake & growth in vitro by

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mycorrhizal fungi. Can. J. Bot. 62, 647-652

Rogers S.O., Rehner S., Bledsoe C., MuellerG.J. & Ammirati J.F. (1989) Extraction of DNA

from fresh, dried & lyophilized fungus tissue forribosomal DNA hybridizations. Can. J. Bot. 67,1235-1243

Rygiewicz P.T. & Bledsoe C.S. (1984) Mycorrhi-zal effects on potassium fluxes by Northwestconiferous seedlings. Plant Physiol. 76, 918-923

Rygiewicz P.T., Bledsoe C.S. & Zasoski R.J.(1984a) Effects of ectomycorrhizae & solutionpH on 15N ammonium uptake by coniferousseedlings. Can. J. For. Res. 14, 885-892

Rygiewicz P.T, Bledsoe C.S.&

Zasoski R.J.(1984b) Effects of ectomycorrhizae and solutionpH on 15N nitrate uptake by coniferous seed-lings. Can. J. For. Res. 14, 893-899

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