Effect Infection Hypomyces Cucurbitae Apparent Free Space, Cell

Plant Physiol. (1968) 43, 1666-1672

Effect of Infection by Hypomyces solani f. sp. Cucurbitaeon Apparent Free Space, Cell Membrane Permeability,

and Respiration of Squash HypocotylsiJoseph G. Hancock

Department of Plant Pathology, University of California, Berkeley, California 94720

Received May 29, 1968.

Abstract. Initial symptoms and increases in respiration, apparent free space, and rate ofleakage of amino acids occurred concomitantly in squash (Cucurbita maxima Dcne) hypocotylsinfeoted by Hypomyces solani f. sp. cucurbitae Snyd. and Hans. Young, rapidly expandinglesions had greater respiratory rates and apparent free space than comparable tissues fromhealthy plants.

Hypocotyl tissues a,bove (145 mm) lesions possessed greater endogenous respira,torv rates(2-3 times) and lower respiratory quotients than similar tissues from 'healthy plants. But no

differences were found in membrane permeability to nonelectrolytes and water and in apparentfree space between cells above lesions and healthy hypocotyls.

Hiost cells contiguous to fungal hyphae at lesion margins were completely permeable tosolutes and failed to accumulate neutral red or exhibit cyclosis.

Clues to mechanisms of pathogenicity of plantpathogenic fungi and bacteria may be obtained fromearly physiological and biochemical symptoms.Though an indirect approach to studies on physiologyof pathogen!icitv must be applied cautiously, studiesoIn early stages of infection can provide new in-formation onl processes peculiar to pathogenesis.

Changes in cell membrane permeability are anearly symptom in many plant diseases (9,13, 22, 24,25). Increases in host membrane permeability aredetectable prior to symptom *expression during in-fection by Rhizoctonia solani Kuhn (13) and wellin advance of lesions in other diseases (21, 22, 24).WVhen cell membranes are destroyed, the normalfunctioning of cells is impossible. In addition toserving vital physiological functions, intact cell mem-branes are essential for sulbcellular organization.Pathological processes which irreparably damage cellmemnbranes may lead to cell deatlh, while subtlechaniges in differential permeability maxy modify hostmetaboltismni.

Physiological characteristics of senescing aniddiseased planit tissues are often similar, and (lirectcomparisons may be instructive. For examiple. se-quential changes in permeability and respiration infrtuit during the climacteric (16) and in Hclm iniitho-sporiuimat toxin-treated tissues ( 24) mav- he cautsa-tivrelv related. Studies with different types of senies-cing tissues indicate that a variety of relationship)sis possible (1. i5, 115). Likew-ise, much !variability

1 Supporte(d in part by Cancer Researclh Fundls of thet'Uiiversitx of California.

can be expected with plant diseases and each (liseaseshould be considered individually.

Hyponyces (Fusarimn) solani f. sp. cuicutrbitacSnyd. and Hans., race 1, causes lesions in the baseof hypocotyls of many members of the Cuccurbitaceac.The disease is severe, and seedling infection utsuallyleads to the premature death of the host. Thoughthe pathogenic and genetic nature of H. solani f. sp.citcicrbitac has been studied extensively (6, 7,23),there is little information on mechanisms of patho-genicitv and the 1)hvsiology of host-parasite inter-actions (10, 11).

The object of the present study was to examinethe effects of Hypontyces-infection on host membranepermeability and to evaluate the relationship betweenmlembrane permeability and otlher host physiologicalactivities during pathogenesis.

Materials and Methods

The clone (Cu-56) of Hyponmyces solani f. sp.cutcutrbitac, race 1, used in this study was maintainedas described previously (10).

Infected squash (Cutcutrbita mnaximta Dcne "PinkBlanana') plants were obtained in the greenhouseby planting seeds in flats containinilg field soil andcoarse sand in a 3 :1 proportion and the pathogen(chlcamvydospores), Healthy plants were grown incomparable noninfested soils. Plants infected in thiw-ay were titilized to stutdx chalnges in respirationand permeability in lesions and in hypocotyl tissuesadjacent to lesions. Lesions apl)leared at the baseof hvpocotyls about 5 to 7 days after seedling emer-gence. Plants with discrete lesions (ca. 1 cnm in

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IIANCOCK-EFFEICT OF INFECTION ON RElSPIRATION AND P'ERMEAB3ILITY1

length) at the base of hypocotyls were selected asexperimental material. Healthy plants of compar-able age were used as controls. MIost studies ofrespiration and permeability were made with hypo-cotyl tissues from healthy plants and from lesiontissues and adjacent tissues (healthy in appearance)1 to 5 mm and 5 to 9 mm above lesions.

Because of difficulties in predicting initial lesionsites in greenhouse tests, another inoculation schemewas used. Young seedlings (2 days old) weredipped in suspensions of macroconidia (6 X 10'!ml), arranged horizontally in large petri dishes onmoist filter paper, and incubated in the laboratoryin indirect sunlight. Control plalnts were dippedin water. Initial lesion development was more uni-form over the hypocotyl surface than that obtainedin greenhouse inoculations and, thus, more useful instudies on early permeability changes. Symptonisoccurred in 2.5 to 3.0 days.

Respiration. Endogenous respiration rates anidrespiratory quotients (R Q) were determined at250 by conventional manometric procedures with aGilson Differential Respirometer (26). Reactionflasks, wrapped with aluminum foil to exclude light,contained 3 ml 0.05 M potassiumll phosphate buffer,pH 6.5, and 40 1-mm-thick hypocotyl cross sections(ca. 0.4 g fr wt) with or without 0.3 ml 20 % KOHin the center well. Respiration rates are expressedas /l 09 absorbed/hr/mg dry weight (Qo.,).

Dissimilation of 14C-gln1cose. Comparisons ofamounts of 14CO9 evolved from glucose-U-14C weremade with tissues above lesions and healthy hvpo-cotyls. Because of differences in respiration ratesand the rate of oxidation of 14C-glucose to 14CO.,the C,:C, ratios of these types of tissues were deter-mined, taking into account the rates of substratecycling discussed by Daly et al. (4). Healthy tissueswere normally incubated with 14C-glucose labeled inthe C-l or C-6 position for twice as long as diseasedtissues. In this way qualitative differences in glu-cose dissimilation in differenit tissues could be ex-amined.

Determination of Apparent Frce Space. A dilu-tion procedure was used to determine the percentageof tissue volume composing the apparent free space(16). Weighed batches of 20 1-mm-thick crosssections of squash hypocotyls (200-300 mg fr wt)were incuibated in 0.5 ml isotope solutions in 2 mlshell vials (12 X 35 mm) for 30 min at 300. Solu-tiolns were buffered at pH 6.5 with 0.03 M phosphatebuffer and contained 0.1 M mannitol-1l-4C (2 kc/ml).After 30 min, duplicate samples (20 ,ul) of theambient solution were removed for radioactivitymeasuremenits. The length of time (min) to achieve20,000 counts was noted. Calculations of percentageapparent free space were made as described bySacher ( 16).

Amino Acid Leakage. Batches of 10 1-mm-thickhypocotyl cross sections were placed in cheeseclothbags and rinsed briefly with distilled water. T:hebags were blotted and placed in 50 ml beakers con-

taining il nil 0.5 AI glycerol (2). The beakerswere shaken in a water bath at 250. Samples of thebathing s-olution (1 ml) were removed at 10, 70,and 130 min for amino acid determinations. At theend of the incubation period, absolute ethanol(35 ml) was added to eaclh sample. Samples wereextracted for 2 hours at 700. Eaclh ethanol extractwas diluted to 100 ml and aliquots wN-ere taken foramino acid measurements (14). WVith these data,total original amounts of amino acids can be esti-mated for each samrple. Since levels of amino acids(and other solutes) fluctuate greatly in plant tissuesand rates of diffusion obey Fick's laws, it is neces-sary to base leakage data on endogenous levels inorder to avoid serious errors (2) ; this is partictu-larlv imiportant w-heni comparing healthy and diseasedtissues.

Permieatioii of I'C-urea Inito Osnwtotic VoluItes.As shown in figuire 1, diffusion equilibria of 14C-mannitol or 14C-urea occurred between bathing solu-tions and the entire hypocotyl tissue volume within30 min when tissues were preheated for 5 min at65°. On the other lhand, 14C-mannitol reached adiffusion equilibriumiwith a restricted portion (ap-parent free space) of the total tissue volume while1-C-urea passed into the mannitol apparent free spaceand then through a diffusion barrier at a reducedrate but in accordance with Fick's Lawv (fig 1).This information suggests that 14C-utrea permeatesthrouglh cell plasima membranes inito the osmoticv"olumes while '4C-mannitol passes these membranesat negligible rates. Thus, studies on the permeationof 14C-urea into comparable tissues could yield usefuldata onl relative membrane permeability. Experi-ments were performed identically to those done onapparent free space except bathing solutions con-tained 0.1 M '4C-urea (2 uc/ml) rather than 0.1 AI'4C-mannitol.

Plasmomietric Determination of Passivc Perme-ability. Cell membrane permeability to specific

w

Exvo

.t

E

~o

aC)

gj,

0

Incubation Time (min)FIG. 1. Permeationi of 14C-mannitol anid '4C-urea

into squash hypocotyl sections. Insert sho- s diffusionequilibrium occurred within 30 mim when sections thatwere heated at 650 for 5 min were batlhed in eitlhersolution. The vertical bars represeint the ranige.

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solutes (S) can be deternmined from the perimieabilitvconstanit (K) taken from the modified Fick equa-tion (20):

dS

dtKA (AC) 9

where S is the amlount of permeating solute, K, thepermeability constant, .4, the surface area of theprotoplast, A(', the difference in cellular and extra-cellular conceintrations of 8, and t, is time.

With the plasnmometric method, K can be calcu-lated for individual cells using an integrated forniof the Fick equation (20). The opportunity tomeasure K values of single host cells at variousdistances from fungal hyphae makes this procedureparticularly attractive in studies of host-parasiterelations.

In measurements of K for urea, an epidermalstrip was plasmolyzed for 15 to 30 min in 1 M ureaand placed in a drop of 1 Mi urea on a cover slipwith the cuticle toward the glass. Then the coverslip was placed on a beltsville watch glass partiallyfilled with 1 M urea in a hanging drop arrangementand the dish placed on a slide on a microscope stage.After a suitable cell with parallel lateral walls waslocated, the length (L) of the protoplast was re-corded during the dilation phase with an eyepiecemicrometer. With this niethod, measuremenits couldbe made for several hr. In most cases L was foundto increase linearly. Calculations of K for ureawere made using aII equatioin (Eq 30) of Stadel-mann (20).

Determinations of K for water were made byplasmolyzing epidermal strips in 1 M urea and fol-lowing the change in L when 1 M urea was replacedby 0.7 M urea. Epidermal strips were placed undercover slips in 1 Ni urea on a microscope slide, suit-

Table I. Rates of 14CO2 Respired and C6:C1 Ratios of1Hypomyces-Affected and Healthy Squash Hypocotyls

Radioactive glucose (G) was diluted in 0.1 M potas-sium phosphate buffer, pH 6.5, and 0.025 M G to giveradioactive solutions of either 2 ,tc/ml (G-1-14C, G-6-'IC) or 4 ,uc/ml (G-U-14C). The 14,CO2 evolved wastrapped directly on planchets in vessels shaken in a waterbath at 280 by a method described by Daly et al. (4).G-U-14'C, G-1-1IC, and G-6- 1C, had specific activitiesof 6.4, 4.4, and 7.0 Jkc/mole, respectively (New Englant(Nuclear Corp., Boston, Mass.).

HealtlivAMea0m

Tis.sue 1-9 mnabove lesionl

M\ean RaiigeRanige14CO,, respired (cprm Per hr per g fr wet)

2145 1900-2390 3870 3290-4450C :C1 ratio

0.45 0.37-0.57 0.33 0.224.43

able cells located, aind 0.7 M urea drawni uniider theedge of the cover slip with filter paper. Measure-nments of cell width and changes in length (L.,-L, )wvere used following statistical rules established bhStadelmnaun (19, E(I 2) in calculatin-g the K forwater.

Results

Respirationt. Endogenous respiration rates (Qo, )were higher in lesions and in tissues above lesionsthan in hypocotyls from healthy plants (fig 2). TheQ02 of diseased hypocotyls was twice the value ofhealthy hypocotyls even 45 mim above lesions (fig 3).Respiratory quotients (R Q) were lower in tissuesabove lesions (0.75) than in those from healthyhypocotyls (0.90).

10-

2 6

.5~~~~~~~~~~~~~~~~~~~~~~

HeoftheLesion. S-9,HeolthLesiorcmfrS,,eS-9,

0 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~3

MMiFli Ab.. Loos, M UM Afrm LesisnFIG. 2. (left) Endogeinous respirationi rates of lealthy and Hvpomvces-infected sunaslh Ix-pocotyls. The vertica!

bars represent the standard errors.FIG. 3. (middle) Endogenous respiration rates in hypocotyl tissues above the margini of lesions. Conitrol i,s respira-

tion found in hypocotyls of healthy planits.FIG. 4. (right) Rate of leakage of amino acids from healthy and Hypomyccs-infected squash hypocotyls. A-minou

acids were measured by the ninhlydrin method of Rosen (14) with glycine as the standard. Amino acid leakage isexpressed as the fraction of total amino acids leaking into bathing solutions per hr (increase in amnoles amrinoacids in bathing solution's per hlr prnoles amitno acids in tissuies at start of experiment).

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Dissimilation of 14C-glucose and C,, :C1 Ratios.As shown in table I, glucose-U-14C was oxidized to'ACO2 twice as rapidly in hypocotyl tissues abovelesions than in comparable ti-zsues from, healthyplants. The C6:C1 ratios were less in tissues abovelesions than in those fronm lhealthy plants in 3 dif-ferent experiments. However, in 1 experiment thehealthy tissue exhibited a C', :C, ratio similar to thatof diseased tissue. Other than documenting thatnormal host metabolisnm is disrupted in diseasedtissue, these results are inconclusive concerningspecific changes in host metabolic pathways.

Leakage of Amino Acids. There was ani accel-erated rate of amino acid leakage only froin lesionsper se. The rate of leakage of amino acids frontissues above lesions and from healthy hypocotvlswere similar (fig 4). In time-course studies (fig5) it was noted that increased amino acid leakageoccurred concomitantly with initial disease symptonms(water soaking). Simlilar studies showed that anincrease in Qo2 of inoculated plants did not occuruntil symptoms appeared.

Apparent Free Space. Since 14Cnmannitol passesintact membranes at negligible rates, an increase inapparent free space represents an increase in "theproportion of cells which has become totally perme-able to solutes by simple diffusion" (16). In lesions.fungal hyphae occtupy a portion of the total tissuevolume and prevent an accurate measurement ofapparent free space. Nevertheless, it is useful tocompare healthy and diseased tissues, and particu-larly fungus-free tissues above lesions with healthytissues.

It is clear from a number of types of experimentswith 1`C-mannitol that there are no significantdifferences in the apparent free space in healthyhypocotyl tissues and tissues adjacent to lesions(fig 6). However, lesions contained a higher per-centage apparent free space than other tissues

studied. Incubation of tissues in radioactive man-nitol solutions up to 150 min failed to demonstratefurther changes in apparent free space. Similarresults were obtained with 'AC-sucrose.

Permeation of 14C-urea Into Tissues. Penetra-tion of 14C-urea into the osmotic volume appeared tobe a passive process. After equilibration with ap-parent free space, a first-order curve is obtainedwhen the percentage of diffusional equilibrium of14C-urea with the tissue volume is plotted as afunction of incubation time; this suggests that labeledsolute permeation obeys Fick's law. Metabolic in-hibitors known to affect active transport, such asNaF (10-3 M) and 2,4-dinitrophenol (5 X 10-5 M)had no effect on solute permeation into tissuesalthough they inhibited respiration 70 % and 98 %.respectively, at the concentrations used. Inhibitorstudies of solute transport are difficult to interpret(12), but these results indicate that energy-dependenttransport processes are not directly involved in themovement of urea across cell membranes.

As shown in figure 7, the rate of permeation of14C-urea into tissues (apparent free space and os-motic volume) was about the same in the healthycontrol tissues and tissues above lesions. Thegreater penetration into lesions probably reflectsmovement into the larger a-pparent iree space.Similar results were obtained with 1'C-H-lycerol.

Uptake and Retention of 14C-glycerol. Thecapacity to retain solutes is a permeability propertyof membranes that can be measured and used toevaluate alterations in membrane properties. Move-ment of 14C-glycerol into osmotic volumes of healthytissues and tissues above lesions was similar whenisotope removal from bathing solutionas was meas-ured. When squash tissues were leached briefly,the isotope in the apparent free sqpace was removed,leaving that contained within the protoplast. Asshown in table II, the amount of 1'C-glycerol re-

-OCt 0 80t r 120 m.c

C -Monnitol l: C -Ureo

* 60 a-60~~~~~~~~~~~~~~~~~~~~~~~~~~60r,D° xwe07s s E 80 0

.elOO -- - -s I2

3~00 Io >

Days after mnacalation 20O2

75~~~~~~~~~~~~~~~~~~~~~~~~a

Healthy Loesio MMAoeLoHealthy Lesaon ,1, 5

.59,

FIG. 5. (left) Increases in rate of amino acid leakage (% of control) and disease indices as a function of timeafter inoculation of sqluash seedlings with HIvpoinyces.

FIG. 6. (middle) Apparent free space in healthy and Hvpomyces-infected sqluash hypocotyls.FIG. 7. (rig,ht) Percentage diffissionlal equilibrium of 14C-urea with sections from healthy and Hvpomyces-

ifetedl sqluash lvpootvls in 60 or 120 nun.

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Table II. Retention of 14C-Glycerol in SquashHypocotyl Tissues

Hypocotyl sections were bathed in 0.1 M glycerol-U-1'C (2 ,uc/ml) in 0.03 M phosphate buffer, pH 6.5.After incubation periods of 2 or 4 hours, the sectionswere rinsed 3 times and leached for 15 min with distilledwater. After leaching, the tissues were rerinsed twice,and incubated overnight with 1 ml 70 % ethanol at 40.Values represent ethanol soluble materials remaining intissues after leaching.

Tissue

HealthyLesionMm above lesion

1-55-9

cpm/g fr wtincubation time

2hr 4hr

125,000 256,00096,000 102,000147000 254.000148,000 268,000

not impaired short distances from the pathogen.In contrast to urea permeation, the water perme-

abilitv of host cells bordering lesions (20-30 cellsfrom hyphae) is greater than that of cells in com-parable tissues from healthy hypocotyls. The valid-ity of this difference is not clear, howx-ever, sincedeplasmolysis often occurred too rapidly to mlakemeasuremenits within the boundaries of ruiles used incalculating K for water. Measurements of K ofcells in epidermal tissue from hypocotyls of healthy,plants were less difficult. Mean (+ SD) K valuesof 5 cells froml tissues of healthy plants and fromthose abo-e lesions were 3.4 + 2.2 X 10)4 and4.9 ± 2.4 X 10-4 cmi/sec, respectively.

Because of problems in measuring deplasmolysisrates, the nmean K value obtained for host cells abovelesions nmay be low. For example, wlen K values(9 cells) fromii measurements within a les- restricted

tained after leaching was about the same in bothhealthy tissues and tissues above lesions. Muchmore isotope was lost during leaclhing of lesiontissues since the plasma membranes lhad lost theirsolute retention capacities.

Permeability Constants. The resuilts of studieson urea permeability with the plasmometric methodare presented in table III. Permeability constants,K, represent mean values of 10 cells, each tested ina separate experiment; this probably accounts forthe large stanidard deviation.

Only host cells contiguous to funigal hyphae orslhort distances from hyphae at lesion margins couldnot be plasmolyzed (fig 8). In some cases hyphaewere observed adjacent to host cells that accumulateda vital stain (neutral red), exhibited cyclosis. andcould be plasmolyzed with 1 al uirea solution-,.Occasionally, plasmolyzed cells close to hyphae burstprematurely during the dilationi phase: this phe-nomenon was not consistent, however.

Host cells within 2 to 10 or 20 to 50 cells fromfungal hyphae possessed permeability constants, K,for urea that did not differ statistically (t 1.06,df=18) from constants for cells in healthy hypo-cotyls. Thus, even when cellular dimensions areconsidered, cell membrane permeability to urea is

Table III. Permeability Constant for Urea inHypomyces-Affected and Healthlw Squash

Hypocotyl CellsValues determined by the plasmometric procedure

(20). A total of 10 cells were measured in each plantmaterial in 10 separate tests.

Mate- No. cells Permeability constant (K)rial from hyphae Mean + SD Range

cm sec-1 cm sec'1HealthY ... 4.6 ± 2.0 X 410-8 1.0-7.2 X 10-8Diseased 2-10 3.8 ± 1.8 X 10-3 1.5-7.4 X 10-8

20-50 3 8 ± 2.3 X 108 1.34.4 X 10-8

FIG. 8. Photomicrograph of squash h-pocotvl epi-dermis from lesioni mlargin. Note relation of host cells(plasniolyzed) to Hypon t' c h)yphae ('arrlocRN- in ad-jaceint dead host cell.

toleranice limit (19) were used in calculationls, a Kvalue of 6.5 ± 2.7 X 10-4 cm/sec was obtained.However, the range of K values for epidermlal ceilsin healthy plants (1.5 - 7.4 X 10-4 cm/sec) wa;not far removed from the range of K v\alues forepidernmal cells above lesions (1.6 - 9.() X 10-4cm/sec).

Changes of water permeability couldI be cauisedby changes in the reflection coefficient (3). Inorder to avoid this problem, comparisons. of the

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Table IV. Water Efflux in Hypomyces-Affected andHealthy Squash Hypocotyl Tissue

Loss of water and solutes from hypocotyl sectionsafter 30 min incubation in 0.44 M mannitol at 300by the method of Glinka and Reinhold (8).

Mean + SDMaterial Water loss Solute loss

mg mgHealthy 132 + 18 43 ± 11Above lesion 140 ± 7 30 + 7

(1-13 mm)

hydraulic permeability in healthy hypocotyls andtissues above lesions were made by the water effluxmethod of Glinka and Reinhold (8). No differencesin water efflux were detected between tissues 1 to13 mm above lesions and those of lhealthy plants(table IV).

Discussion

The cause and effect relationship of the perme-ability-respiration problem has been considered ex-tensively in studies on fruit ripening and senescence

(5, 15, 16). For both processes changes in perme-ability may precede changes in respiration. Becausethe destruction of cellular compartmentation wouldallow respiratory substrates and enzymes to interact,some workers consider increases in membrane per-meability responsible for the increases in respirationassociated with the climacteric of ripening fruit.However, when permeability increases were inducedexperimentally in cantaloupe fruit, a respiratorychange did not occur '(1). Indeed, in other senescingvegetative tissues, increases in permeability may befollowed by a decline in respiration (5). The rea-

sons for differences in respiratory responses to in-creased permeability are vague, but it appears thatthe physiological condition of cells at the time per-

meability is altered determines the nature of second-ary responses.

Durinig early stages of infection, increases inrespiration are detectable concomitantly with initialdisease symptoms (water soaking). Increases inrates of leakage of amino acids and in apparent freespace occur at this time. If there is a cause-effectrelationship between increases in plasma memlbranepermeability and respiration during early stages ofpathogenesis, it cannot be determined from theseresults. Howvever, there is no relationship betweenthe large increases in endogenous respiration foundin host tissues above lesions and solute permeability.Plasma membrane permeability to nonelectrolvtes,such as water, glycerol, mannitol, and urea, and to

a'minio acids, did not change in tissues above lesions.Thus, it is concluded that increases in endogenotisrespiration above lesions cannot be caused by changesin passive permeability characteristics of host plasmiamembranes.

In several diseases caused by facultative parasites,cell permeability is increased at large distances fromthe pathogen. For example, changes in permeabilityoccur well in advance of the pathogen, Helmintho-sporiumn victoriae (24), and it is now suggested thatthe host specific toxin produced by this pathlogenselectively damages host plas,ma membranes (17 ).In sharp contrast to this effect by H. victoriae, Hypo-myces solani f. sp. cucurbitae does not affect mem-brane permeability in host cells except those coIn-tiguous or in close proximity to the hyphae. Mosthost cells in tissues adjacent to lesions plasmolyzeand deplasmolyze in a normal manner, accumulatevital stains and exhibit cyclosis. Permeability coIn-stants of host cells for urea in healthy hypocotylsand tissues adjacent to lesions are of the samemagnitude., These results suggest that potent niem-brane-damaging toxins, such as victorin, are eithernot produced, or not produced in great quaIntity.during pathogenesis by H. solani f. sp. cucurbitac.Host cells 1 to 3 cells from lhyphae at the margin

of young lesions would not plasmolyze. This sug-gests that these cells are freely permeable to solutes.The loss of the differential permeability of thesecells could account for the water soaking at themargin of lesions. The cause of the membranedamage is unknown, but, if fun)gal products aredirectly responsible, they either cannot diffuse rap-idly into adjoining hypocotyl tissues or their activi-ties are greatly reduced by dilution, immobilization.or destruction.

The efflux of water from tissues above lesions(1-13 mm) did not differ from that of controls,although results from plasmometric studies indicatethat water permeability may be increased slightly inliving host cells close to hyphae. Before any con-clusions can be drawn on changes in water perme-ability in host cells close to fungal hyphae, morereliable data are needed.

When the differential permeability of host cellsis destroyed, a diffusional equilibr.ium of cell metabo-lites and inorganic nutrients occurs between thedamaged protoplast and apparent free space. Thisis 1 way in which nutrients are made available tothe pathogen growing in intercellular spaces. Wlhelncell permeability increases well in advance of thepathogen, nutrients diffusing into intercellular spacesmay be utilized to a large extent by saprophyticmicroorganisms. For example, Skou (18) reportedthat common saprophytic microorganisms invadeplant tissues rapidly when cell permeability is in-creased by &-radiation. WVlhen alterations in cellpermeability are limited to host cells close to hvphae,cell metabolites will be utilized more efficientlv bythe pathogen. This conservative relationship mayreflect a higher degree of parasitic specialization(21) of H. solani f. sp. cucurbitae than of otherfacultative parasites, such as Sclerotinia sclerotioruni(Lib.) d By. (21, 22) or RIhi.octonia solanii (13),vWhich cause increases in permeability at large dis-tances from the fungus.

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Acknowledgments

The autlhor is grateful to Drs. W. C. Snyder anidA. R. \Veinlhold for reading the manuscript anid for tlheiradvice

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