Physiological Plant Pathology (1985) 27, 323-334

Activation of superoxide generation and enhancement of resistance against compatible races of Phytophthora infestans in potato plants treated with digitonin

N. DOKE and H. B. CHAI

Plant Patholou Laboratory, Facula of Agriculture, .Nagoaya Universily, Chikusa-ku, Nagova 464, Japan

(Acceptedforpublication July 198.5)

Application of digitonin to the leaf surface of potato plants was demonstrated to activate O,- generation of the leaf tissues as determined by extracellular cytochrome c reduction which was inhibited by superoxide dismutase (SOD). Protoplasts prepared from stem shoots of potato enhanced NADPH-dependent reduction of extracellular cytochrome c immediately after being treated with digitonin. The reduction was also inhibited by SOD. The similar activation of SOD- sensitive cytochrome c reducing activity was observed in leaves or protoplasts ofsome other plants treated with digitonin.

Potato leaf tissues pre-treated with digitonin by application to the upper or lower surfaces, or through the petiole, were protected from disease caused by infection with compatible races of Phytophthora infeestans. Treatment ofthe wound surface ofpotato tuber tissue with digitonin induced the generation ofthe superoxide anion and, at the same time, sporangial germination, appressorial formation and invasion by P. infestans was greatly reduced. This effect was partially negated by the presence ofSOD in the inoculum and seemed not to depend on antifungal compounds.

These results suggest that plant tissues possess an O,- generating NADPH oxidase which is activated by digitonin and that its activation in potato may contribute to an enhanced resistance against attack by compatible races ofP. infestans at pre- or post-infection stages.

INTRODUCTION

Wounded potato tuber tissues have been found to activate a superoxide (O,-) generating system almost immediately after penetration of the cells by incompatible, but not after penetration by compatible, races of Phytophthora i&tans [5j. The activated system was found to be an 0 s- generating NADPH oxidase by using potato tuber protoplasts reacting to hyphal-wall components of the fungus [6J or a membrane- rich fraction isolated from tissues activated by infection with incompatible fungi [7j. From these findings, the activation of an O,- generation system was interpreted as a biochemical reaction which was activated following recognition by the cells of incom- patible fungi, and which triggered the subsequent dynamic resistance reactions [5J.

In potato leaves inoculated with P. infestans, an activation of O,- generation was found to occur before penetration by both compatible and incompatible races, with further 0, - generation following penetration by incompatible, but not by compatible, races [Z]. The former reaction was also found to be activated in leaf tissues by germi-

Abbreviations used in text: SOD, superoxide dismutase.

0048-4059/85/060323+12$03.00/0 0 1985 Academic Press Inc. (London) Limited

324 N. Doke and H. B. Chai

nation fluid of P. infestans cystospores [Z]. The pre-inoculation treatment of potato leaves with germination fluid has been shown to reduce susceptibility to compatible races [Z]. Thus, O,- generation in potato tissues seems to be involved in self-defence against P. infestans, before or after penetration. If this is true, compounds, which activate the O,- generating system in potato tissues, would be expected to act as a protectant of the potato plants against the fungus.

In a test screening of certain compounds, we found that digitonin, a steroid glyco- alkaloid from Digitulispurpurea L., activated the 0 z- generation system of potato plant tissues [S]. In the present paper, we describe some aspects of O,- generation acti- vated by digitonin in potato as well as in some other plants, and demonstrate that pre-inoculation treatment of potato tissues with digitonin reduced their susceptibility towards compatible races of P. infestans. A brief report of these studies has been published elsewhere [S].

MATERIALS AND METHODS

Plants The following plants were used, potato (Solanum tuberosum L. cv. Irish Cobbler and an interspecific hybrid between S. tuberosum and S. demissum L. cv. Rishiri), tomato (Lycopersicon esculentum Mill, cv. Fukuju-P-go), sweet pepper (Capsicum frutescense L. cv. New Ace), tobacco (Nicotiana tabacum L. cv. Xanthi nc.) kidney bean (Phaseolus vulgaris cv. Kairyo-Otebo), soybean (Glycine max Merr., cv. Raiko-Edamame), pea (Pisum sat&m L., cv. Sanukinagasaya), cowpea (Dolichos sesquipendalis Wight, cv. Kurodane- Sanjaku).

For leaf material, fully developed leaves of solanaceous plants (leaves 4 or 5 from top of plants) grown in pots for 2-3 months after planting in a greenhouse were used. The first true leaves of leguminous plants grown in a greenhouse were used before the second true leaves were well developed. Protoplasts were prepared from shoots of potato tubers grown in vermiculite in the dark at 20 “C for l-2 weeks, hypocotyls of leguminous plants grown in vermiculite in the dark at 25 ‘C, and green fruits of sweet pepper and tomato. Potato tuber tissue discs (20 mm in diameter, 2 mm thick) pre- pared as described elsewhere [4], and aged in the air at 20 “C for 18 h after slicing, were also used.

Preparation of protoplasts Shoot tips (l-2 mm thick, 5-10 mm long) prepared from each plant, were washed in a medium consisting of 0.05 M potassium phosphate buffer (pH 5.5), 0.6 M mannitol, 1 mM MgCl,, 1 mM CaCl, and 0.01% /%mercaptoethanol; 2 g of the tips were suspended in 15 ml of the medium with 2% cellulase (Onozuka R-10) and 0.05% pectolyase Y-23 (Seiisshin Pharmaceutical Co. Ltd.) at 25 “C for 3-4 h with slow reciprocal shaking (50 times min- ’ ). The protoplasts released in the medium were isolated as precipitates by centrifugation at approx IOOg for 2 min after the total incubation medium had been filtered through a mesh. The precipitated protoplasts were resuspended in 0.05 M potassium phosphate buffer (pH 7.8) containing O-6 M

mannitol and 0.1 mM EDTA, and washed three times by centrifugation. This resulted in the enzyme medium being diluted approx. 1000 times.

Superoxide generation with digitonin

Fungi and inoculation

325

Phytophthora infestans (Mont) de Bary race 0 and race I .2.3.4 were used for pathogenic fungi virulent against potato cv. Irish Cobbler (r) and Rishiri (R,), respectively. The former race was also used as an incompatible race to the latter cv. Preparation of a zoospore suspension as inoculum was carried out as described elsewhere [I]. A zoospore suspension, adjusted to a concentration of 3 or 80 x 1 O4 ml - ‘, was spread on the upper surface of leaves or tissue discs of potato, respectively. These inoculated tissues were incubated in a moist chamber in the light at approx. 3000 lux at 20 “C for 14 h a day or in the dark at 20 “C.

Treatment with digitonin A digitonin solution containing 0.5% ethanol at 100 ppm (81 PM) was applied to the entire upper or lower surface of leaf tissues, on the upper surface of potato tuber discs, or through the leaf petiole of potato plants.

A digitonin solution, dissolved in the medium used for protoplasts suspension, was added to the assay system for O,- g eneration by protoplasts, at a final concentration of 100 ppm. As the control, O*5o/o ethanol was used.

Assay for 0, - generation 0, - generating activity of leaf tissues, potato tuber discs or protoplasts was assayed by determining the reduction of extracellular cytochrome c, which was inhibited by SOD (100 pg ml-‘), as previously described [5, s]. In the former two cases, optical density at 550 and 540 nm of an assay mixture consisting of 0.05 M potassium phosphate buffer (pH 7.8), 20 C(M cytochrome c (Type VI from horse heart, Sigma Chemical Co.) and 0.1 mM EDTA was spectrophotometrically assayed in a 10 mm cell, using a Hitachi double beam spectrophotometer (Type 200-20) for 10 min, after adding five leaf or tuber discs into the assay mixture (3 ml) with or without SOD (100 pg ml-’ at final concentration). The protoplast reaction was started by adding digitonin to a protoplast suspension consisting of 0.05 M potassium phosphate buffer (pH 7*8), 20 pM cyto- chrome c, 10 PM NADPH, 0.6 M mannitol and 0.1 mM EDTA, and stopped periodically by adding &-ethylmaleimide (10 mM at final concentration). After centrifugation of the protoplast suspension at approx. 1OOg for 2 min, the supernatant was subjected to the optical density assay at 550 and 540 nm. As a control, the protoplast suspension was treated with SOD (100 pg ml-‘) or without digitonin or NADPH. Certain inhibitors were also tested.

Microscopic observation Cystospores, germinated cystospores, germinated cystospores forming appressoria, and invading fungi on the upper surface of potato tuber discs were observed directly under a light microscope after making thin sections with a razor blade.

Determination of antifungal compounds Phytoalexin (rishitin) concentration was determined using gas liquid chromatography after extraction from potato tuber discs as described previously [9]. Bioautography for antifungal compounds was carried out by spraying spore suspensions of Cladosporium

filvum on silica plates according to the method of Allen [I].

326

RESULTS

N. Doke and H. B. Chai

Activation of 0, - generation in plant leaf tissues by digitonin Leaf discs (10 mm in diameter) of potato, tobacco, sweet pepper and bean, treated with digitonin, showed detectable extracellular cytochrome c-reducing activity within 1 h of treatment and activity increased with further incubation time (Fig. 1). In potato and bean leaves, the activity reached a maximum 2-3 h after treatment, but continued until at least 5 h after treatment. In the assay medium with SOD, the activity in each plant tissue was similar to that in untreated material, suggesting that cytochrome c reduction may be due to O,- generated by the leaf tissues. In other plant leaf samples (soybean, pea, cowpea and tomato), the activity was assayed 4 h after treatment with digitonin. Each case showed enhanced cytochrome c-reducing activity which was inhibited in the presence of SOD (data not shown). Heat-denatured SOD did not cause inhibition.

Potato

t.

Beon

;r”

?#zkt 2 4

Tobacco

ii

& 2 4

Pepper

i: A----;(:

2 4 Time ofter treotment (h)

FIG. 1. Time course of 0, - generating activity in plant leaf tissues following application of digitonin solution onto the upper surfaces. The activity was assayed by measuring spectro- photometrically the reduction of extracellular cytochrome E in an assay medium with or without superoxide dismutase as described in Materials and Methods. Thirty microlitres of 1OOppm digitonin were applied per leaf disc (10 mm in diameter). 0, Treated tissue, assayed without SOD; X, treated tissues, assayed with SOD; 0, untreated tissues, assayed without SOD. Each value represents the average of three experiments with standard deviations.

Activation of 0, - generation in wounded potato tuber discs by digitonin The potato tuber discs aged for 18 h after slicing (10 mm in diameter, 2 mm thick) and treatment with digitonin, had detectable extracellular cytochrome c-reducing activity from 1 h after treatment (Fig. 2). The enhanced activity reached a maximum about 4 h after treatment, and remained constant for at least 8 h after treatment. The enhanced activity was scarcely detected in the assay mixture containing SOD (Fig. 2). Untreated discs showed little reducing activity.

Superoxide generation with digitonin

x T. 0 I 2 3 4 5 6

Time after treatment (h)

327

FIG. 2. Time course of O,- generating activity in potato tuber discs following application of digitonin solution onto the upper surface. The activity was assayed as for Fig. 1. Fifty microlitres of 100 ppm digitonin were applied per disc (10 mm in diameter, 2 mm thick). 0, Treated discs, assayed without SOD; X, treated discs, assayed with SOD; 0, untreated discs, assayed without SOD. Each value represents the average of three experiments with standard deviations.

0, - generation in protoplasts treated with digitonin In protoplast suspensions from tested plant tissues (potato, soybean, pea, sweet pepper, bean and tomato) extracellular cytochrome c reduction was found to be activated by digitonin in the presence of NADPH (Table 1). T ime course analysis of the activity in potato protoplasts showed an almost linear activation feature with time following treat- ment with digitonin, without a lag phase (Fig. 3). In this assay the addition of SOD to the reaction mixture 2 min after treatment stopped further reduction of cytochrome c. In the assay medium without NADPH, little activation by digitonin was detected

TABLE 1

Digitonin-activated 0, - g enerating activity ofpmtoplastsfrom various plant tissues

Plants Tissues Activitya

pmoles 10 min-’ 100 protoplasts-‘)

Potato Tomato Sweet pepper Bean Pea Soybean

Shoot Green fruit Green fruit Hypocotyl Hypocotyl Hypocotyl

2.0+0.2 1.3kO.5 0.8kO.3 2.1 kO.7 0.9kO.5 2.7kO.7

‘Activity was assayed by spectrophotometrical determination ofextracellular cytochrome c reduced from 5 to 15 min after digitonin treatment. In each experiment, control without treat- ment with digitonin showed no detectable value of reduced cytochrome c and protoplasts treated with digitonin in the presence of SOD (100 pg ml-‘) revealed a negligible value of reduced cytochrome t. Each value represents the average of five determinations.

328 N. Doke and H. B. Chai

2 4 6 8 IO 12 14 Time ofter treatment (men 1

FIG. 3. Time course of O,- generating activity in protoplasts from potato shoots following treatment with digitonin. The activity was spectrophotometrically assayed by determining extracellular cytochrome c reduction in protoplast suspension as described in the Materials and Methods. 0, treated, assayed with NADPH and without SOD; X, assayed with NADPH and SOD; 0, treated, assayed without NADPH and SOD; +, untreated, assayed with NADPH and without SOD. Each value represents the average of 6 determinations in three different experiments with standard deviations.

(Fig. 3). Protoplasts untreated with digitonin revealed little cytochrome c-reducing activity even in the presence of NADPH in the assay medium (Fig. 3). In the activated protoplast suspension, dextran-bound p-chloromercuribenzoic acid, a high molecular weight SH-reagent of approx. 7000 M. W. (1.4 mg-‘) [IO], quickly stopped further reduction of cytochrome c (data not shown). NADP+ and Kethylmaleimide (50 PM

and 1 mM at final concentration, respectively, had a similar effect.

Effect of digitonin treatment on infection by P. infestans in potato leaves. Excised potato leaves were protected from attack by compatible races of P. infestans when they were treated with digitonin on the upper surface 4 h before inoculation [Fig. 4(a) and (b)]. Irish Cobbler leaflets untreated with digitonin were completely destroyed 4 days after inoculation by race 0 while only a few disease spots, which later ceased in their develop- ment, appeared in the treated leaflets [Fig. 4(a)]. Rishiri leaves with both general resistance and R, gene resistance against P. infestans, untreated leaflets showed numer- ous individual disease spots 4 days after inoculation with race 1.2.3.4, while treated leaflets showed only a few tiny disease spots [Fig. 4(b)].

FIG. 4. Protection of potato leaves from late blight disease caused by Phytophthora infestans, by pre-inoculation treatment with digitonin.(a) and (b): digitonin solution at 100 ppm was applied to the entire upper surface of each leaflet (right leaves) or 0.5% ethanol solution used for dissolv- ing digitonin was applied (left leaves); 4 h later, the surface of each left leaflet of the leaves was inoculated with a zoospore suspension (3 x IO4 ml-‘) of compatible races (race 0 or race 1.2.3.4. to Irish Cobbler (A) or Rishiri (B), respectively). c: right and left leaves (Irish Cobbler) were infiltrated with digitonin and 0.5% ethanol solution through the petioles for 4 h, followed by inoculation with race 0 on each left leaflet of the leaves. All photographs were taken 4 days after inoculation.

leroxide generation with digitonin

330 N. Doke and H. B. Chai

According to the number of initial disease spots counted 3 days after inoculation, Irish Cobbler and Rishiri leaf kissues, treated with digitonin received 92 and 83% pro- tection, respectively, from infection with the compatible race (Table 2). In the leaves of Irish Cobbler and Rishiri treated with digitonin on their lower surfaces 4 h before inoculation, the protection was 51 and 49%, respectively (Table 2). Pre-inoculation treatment of leaves with digitonin through infiltration via the petiole for 4 h also pro- tected Irish Cobbler and Rishiri by 73 and 69% respectively. In these cases, however, disease symptoms in protected leaves gradually developed with time after inoculation [Fig. 4(c)].

TABLE 2

Effect of pre-inoculation treatment of potato leaf tissues with digitonin on infection by a compatible race of Phytophthora infestans”

No. ofdisease spots per cm2 b

Application Untreated Treated Protection (%)

On upper surface Irish Cobbler (r) Rishiri (R,)

On lower surface Irish Cobbler Rishiri

Through petiole Irish Cobbler Rishiri

19*5 2&l 92 .22+4 4fl 83

22+4 11+3 51 17*4 9+3 49

15*6 4*2 73 19*5 6*3 69

‘Zoospore suspension of race 1.2.3.4 (100 ~1) at 3 x 104ml-’ was applied to upper surfaces of each leaflet 4 h after treatment with digitonin (100 ppm).

bCounted 3 days after inoculation. Each value represents the average of 12-16 leaflets + SD.

Infection behaviour of P. infestans onpotato tuber discs treated with digitonin. Potato tuber discs (Rishiri) were treated with digitonin, followed by inoculation with Race 1.2.3.4. 4 h later. On the untreated control discs, most cystospores had germinated 3 h after inoculation, while a great reduction in cystospore germination was found on the treated discs (Table 3). Most of the germinated cystospores on the untreated discs formed appressoria and had successfully penetrated by 7 h after inoculation (Table 3). On the treated discs, however, only about 68% of germinated cystospores formed appressoria and only 14% of these penetrated (15 and 3% of total cystospores, respectively) (Table 3). Many ungerminated cystospores on treated tissues appeared to be broken and showed irregular shapes.

When a zoospore suspension with SOD was added to the treated tissues, significant recoveries of the rate of germination, appressoria formation and penetration were

Superoxide generation with digitonin

TABLE 3

Effect of pre-treatment of wounded potato tuber tissues with digitonin on the behaviour o~zoospore~ oj Phytophthora infestans on the treated surface

331

Fungal hehaviour ( y0 of total spores)’

Time after

Inoculation (h)

Treated with water Treated with digitoninb

Appressorium Appressorium Germination formation Penetration Germination formation Penetration

3 95 67 55 21 10 2 5 97 90 80 20 15 3 7 97 95 88 22 15 3

“About 200-300 spores in total were observed in each sample from three different experiments.

‘Tissue discs (Rishiri, R,), on the upper surface of which digitonin at 100 ppm was added, were inoculated with zoospore suspension (race 1.2.3.4) at 8 x 10s ml-’ 4 h after treatment.

100

% 50

l(b)

! W D D+SOD W D

Fro 5. Behaviour of Phytophthora infesttans on potato tuber discs pre-treated with digitonin. (a) Germination of cystospores ( q ), germinated cystospores forming appressoria ( q ) and germi- nated spores penetrating into cells ( n ) on the discs treated with digitonin (D) or with 0.5% ethanol (W) 5 h after inoculation. D+SOD, discs treated with digitonin were inoculated with a zoospore suspension together with SOD (100 pg ml-t). Zoospore concentration in inoculum: 8 x 10s ml-‘. (b) Germinated cytospores in 100 ppm digitonin solution 24 h after incubation of zoospores. W: 0.596 ethanol solution. D: ditigonin solution. Each value represents the average of three experiments, each of which includes about 300 observations on spores. Standard deviations are shown by bars.

observed (Fig. 5). The zoospores suspended in digitonin solution at the concentration added to tissues (100 ppm) apparently showed no effect on encystment of zoospores and germination of cystopores (Fig. 5).

Elicitation of antifungal compounds in potato tuber ti.ssue.s by digitonin. Treatment of potato tuber discs (Rishiri) with digitonin at 100ppm caused no visible damage to tissues

332 N. Doke and H. 6. Chai

within observed times (3 days after treatment). The treated tissues yielded no detect- able rishitin, a major phytoalexin of potato, from 1 to 3 days after treatment as determined by gas liquid chromatography and by bioassay (data not shown).

Untreated discs inoculated with incompatible (race 0) and compatible (race 1.2.3.4) races produced, respectively, a great and a small amount ofrishitin 2 days after inoculation (Table 4). In the digitonin treated discs inoculated with the compatible race, however, a detectable amount of rishitin was produced. In the treated discs inoculated with the incompatible race, less rishitin was produced than in untreated discs inoculated with the same race (Table 4).

TABLET

Phytoalexin (rishitin) accumulation in potato tuber dim treated with digitonin followed by inoculation with compatible OT incompatible races of Phytophthora infestans

Tissues” Rishitinb

(pgg fresh weight-‘)

Untreated, uninoculated N. D. Treated, uninoculated’ N. D. Treated, uninoculated N. D. Untreated, inoculated with race 1.2.3.4 tr. Treated, inoculated with race 1.2.3.4 8*5 Untreated, inoculated with race 0 18f3 Treated, inoculated with race 0 5*2

‘Tissue discs (20 mm in diameter, 2 mm thick), aged for 18 h after slicing, were treated with digitonin and inoculated with 100 p of a zoospore suspension at 8 x IO5 spores ml-’ 4 h after treatment.

bDetermined 48 h after inoculation. Each value represents the average of six determi- nations in three different experiments f S. D. N. D. =not detected. tr. = trace.

‘Determined 4 h after treatment, just before inoculation.

DISCUSSION

Extracellular cytochrome c-reducing activities of potato leaf and tuber tissues or proto- plasts activated by digitonin, were significantly depressed by SOD added in the assay system (Figs. 1, 2, 3), suggesting that O,- was generated by the tissues or protoplasts. Although a restricted number of tissues of other solanaceous and leguminous plants were used in the present experiments, observations of a similar activation in these plants suggests that many plant cells may generate 0, - in response to digitonin (Fig. 1, Table 1).

From the results showing that activation of O,- generation in protoplast was dependent on NADPH and inhibited by NADP+ (Fig. 3), the O,- generation in these treated plants may be catalyzed by an NADPH oxidase. Previous experiments sug- gested that activation of O,- generation in potato tuber tissues or its protoplasts by infection or treatment with hyphal-wall components also depended on NADPH oxi- dation [q. The reason why protoplast systems needed exogenous NADPH for the

Superoxide generation with digitonin 333

activation of 0 2- generation may be because of reductions in the NADPH-generating metabolism, such as the pentose phosphate pathway.

Digitonin is a steroid saponin, that can combine with sterols and cause toxic or physiological changes in plants [12,13]. The concentration of digitonin used in the present experiment (100 ppm = 81 PM) was not enough to cause any visible effect on the plant tissues, and was assumed not to be toxic. In our preliminary experiments, digitonin at concentrations higher than 1 mM did show a toxic effect on plant leaf tissues, sometimes causing tissue collapse. Excess generation of 0, - in the tissues may cause some catastrophic changes in metabolism in the tissues, because O,- and the derivative superoxide (*OH, H,O, or ‘0,) are highly reactive [II]. The plant tissues treated with 100 ppm digitonin presumably escape from the toxicity of superoxides by scavenging it with endogenous SOD, peroxidase and catalase.

The infection behaviour of P. infestans was found to be greatly disturbed in the wounded tuber tissues generating O,-, following treatment with digitonin, although no direct effects on the encystment of zoospores or germination of cystospores were evident (Fig. 5). Digitonin has been known to have an antibiotic action on some fungi, but not on PJthiaceae without sterols [12, II]. The disturbance of fungal morphogenesis in the course of infection seemed to be at least partly due to a direct effect of O,- or the derivative superoxides since co-existence of exogenous SOD in the inoculum partially alleviated the effect ofdigitonin (Fig. 5). However, it is also possible that some plant pro- cess that is activated in plant tissues by O,- generation may affect fungal behaviour. The protection of potato leaves from infection by compatible races, by digitonin treat- ments, is thought to be due to the effect of generated O,- on fungal infection that is found in wounded tuber tissues.

A few fungi successfully invaded leaf tissues treated with digitonin and caused diseased spots, although development of these eventually ceased (Fig. 4). In tuber discs treated with digitonin, the few fungi that invaded the tissue finally ceased growing with the appearance of hypersensitive-like necrosis. These phenomena suggested that some antifungal principle may be produced in response to compatible races. The tuber discs treated with digitonin at 100 ppm produced no detectable antifungal compounds including the known phytoalexin rishitin. However, the compatible race elicited phytoalexin accumulation in digitonin-treated discs to some extent, but not in untreated discs (Table 4). The incompatible race, on the other hand, elicited less phytoalexin accumulation in the treated than in the untreated discs. Thus, these phenomena may be due to the occurrence of an incompatible interaction response to the invading compatible race and the reduction of cells responding to the incompatible race in digitonin treated discs.

The mechanism of elicitation of the O,- generating system by digitonin in plant tissues remains to be elucidated. It is interesting, however, to note that superoxide generating NADPH oxidase in animal granulocytes is also activated by digitonin [3]. Digitonin can be used as a model compound for determining the mechanism of acti- vation of 0, - generating NADPH oxidase caused by infection [5, 4. The present experiments also suggested that the activation of O,- generating NADPH oxidase in plant tissues may contribute to the protection of plants from infection by pathogens. Some other chemicals capable of activating O,- generation may be worth testing for similar plant protection.

334 N. Doke and H. 6. Chai

We are grateful to Mr Y. Nishibe, Hokkaido Agricultural Experimental Station for generously supplying potato seeds. This work was partly supported by a Grant-in-Aid of the Ministry of Education, Science and Culture, Japan (58560044).

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6. DOKE, N. (1983). Generation of superoxide anion by potato tuber protoplasts during the hypersensitive response to hyphal wall components of Phytophthora infestans and specific inhibition of the reaction by suppressors of hypersensitivity. Physiological Plant Pathology 23,358-369.

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Il. RABINOWITCH, H. D. & FRIDOVICH, I. (1983). Superoxide radicals, superoxide dismutases and oxygen toxicity in plants. Phytochemistry and Phytobiology 37,679-690.

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13. VENDRIG, J. C. (1964). Growth regulating activity ofsome saponins. Nature 203, 1301-1302. 14. WATERS B. (1968). The antibiotic action ofsaponin III. Saponins as plant fungistatic compounds. Planta

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