Kinetics of phytoalexin accumulation in potato tubers of different genotypes infected with Erwinia carotovora ssp Atroseptica

Physiological

Molecular

and

Plant

Pathology

(1994)

335

44, 335-347

Kinetics of phytoalexin accumulation in potato tubers of different genotypes infected with Erwinia carotovora ssp a trosep tica ~.HILDENBRAND In.rtiluljiir

Chemische

Accepted

March

and H. NINNEMANN Pjanzenpllysiologie,

CorrenrstraJe

41, 72076

Ebingen,

Federal

Republic

of Germany

1994

The time-courses of rotting and phytoalexin accumulation in tuber soft rot tissue of two cultivars of Solarium fuberosum subspecies fuberosum were examined from l-7 days after inoculation with Envinia carotooora ssp alroseplica. In extracts of infected tissue, rishitin, solavetivone, phytuberin, hydroxysolavetivone, phytuberol, dehydroxyphytuberol, solanascone and various fatty acid ethyl esters were detected. This paper describes a new and convenient method ofextracting phytoalexins and analysing complex extracts with fused silica capillary gas chromatography. Low concentrations of phytoalexins were correlated with extensive rotting. Thus, tubers incubated anaerobically rotted rapidly and extensively and no phytoalexins were detected. When tubers were incubated aerobically, stored mature tubers accumulated considerable amounts of rishitin and smaller amounts ofsolavetivone and phytuberin, and exhibited less extensive rotting. In the tubers of cultivar Grata, rishitin accumulated more rapidly than in those of cv. Irmgard, but the differences in susceptibility were not significant. Aerobically incubated immature unstored tubers rotted extensively and no phytoalexins were found in the rot, which demonstrated the influence of physiological age on the resistance. The selected accessions ofSolanum fuberosum spp andigena showed the most pronounced correlation between high concentrations of phytoalexins in the tissue and low susceptibility.

INTRODUCTION

The bacterium Erwinia carotovora subspeciesatroseptica (van Hall) Dye is the main cause of tuber soft rot and stem rotting of potato, e.g. blackleg, in temperate climates. After infection with E. carotovora ssp atroseptica sesquiterpenoid phytoalexins were found in rotten tuber tissue [19]. Phytoalexins are of interest because their production by the potato tissue may contribute to resistance to bacterial or fungal infections [23,31]. Some of the phytoalexins have antibacterial and antifungal activities [22,31]. In the system Solanum tuberosum/E. carotovora no kinetic data have been published on the speed of accumulation of phytoalexins within the first few days of infection. Thus we examined the phytoalexin accumulation in potato soft rot tissueof two German potato cultivars and two accessionsof S. tuberosum sspandigena Juz. et Buk. over a period of l-7 days after infection. The influences of tuber maturity and physiological condition on susceptibility to soft rot and the accumulation of phytoalexins were compared. We report the occurrence of rishitin, a steroisomer of rishitin, phytuberin, solavetivone, hydroxysolavetivone, solanascone,phytuberol and dehydroxyphytuberol 0885-5765/94/050335+

I3 $08.00/O

0

1994 Academic

Press Limited

S. Hildenbrand

336

and H. Ninnemann

in extracts of infected potato tisstie. The present paper describes a new, convenient method of extracting phytoalexins and a new method of analysing complex extracts using fused silica capillary gas chromatography [22] in a sensitive, precise and reproducible manner. MATERIAL

AND METHODS

Tubers

Tubers of two German cultivars of S. tuberosum ssptuberosum L. (cvs Grata and Irmgard) were produced in the samefield from certified seedtubers. They were either harvested 2 months before maturity and inoculated 1 day later with E. carotovora SSP atroseptica; or they were harvested after maturity and stored for 7 months at 10 “C before inoculation. These cultivars are common commercial genotypes. They were both evaluated with respect to soft rot as possessing intermediate susceptibility: CV. Irmgard is slightly lesssusceptiblethan cv. Grata, although cv. Irmgard is highly susceptible to blackleg. For additional experiments, the German cvs Datura and Hansa were used. In preliminary studies with the “hole inoculation method”, we used commercial tubers of cv. Sieglinde. Two accessionsof S. tuberosum sspandigena, obtained from the International Potato Center (CIP), Peru, CIP-Nos. 703667 and 702299, were used becauseof their resistanceto Erwinia ssp.Both accessionsare resistant to E. chrysanthemi. while accession703667 was also selectedfor resistanceto E. carotovora sspcarotovora and sspatroseptica (Z. Huaman, pers. comm.). These accessionswere grown for 5 months in wintertime in the glasshousewith additional lighting (12-h day) and were stored for 2 months at 5 “C. All stored tubers were held at room temperature for 1 week after harvesting. of E. carotovora sspatroseptica inoculum The bacterium E. carotovora sspatroseptica isolate 30184 was obtained from the German Collection of Microorganisms, (DSM, Braunschweig, FRG). Inoculum for infection experiments wasprepared by multiplication of the bacteria in yeast extract/beefextract medium [containing (g 1-l) yeast extract 1, peptone 5, beef extract 5, sucrose 5; MgSO, .7 H,O 2 mM; pH 7.01. D uring pre-culture the bacteria were grown at 20 “C and 140 r min-’ overnight. The main culture was started with 2 x 10’ bacteria in 70 ml of medium, and the bacteria were shaken for another 17-20 h. After centrifugation at 5lOOg for 12 min and washing twice with 0.15 M phosphate buffer (Na, HPO, .2 H,O/NaH,PO, . H,O, pH 7*0), the bacteria were suspendedin buffer to give 4 X 10’ cellsml-‘. Preparation

and incubation of the tubers The tubers were washedand stored overnight at room temperature. Before inoculation, the tubers were surface-sterilized by vacuum infiltration with sodium hypochlorite (available chlorine O-7 %) containing 0.005 o/oTween 80 for 20 min. After rinsing and vacuum infiltration with sterile distilled water, they were dried in an airflow. Inoculation

” Trough inoculation

method”

Most of the tubers were wounded by making three shallow troughs ‘(1.5 x 3-5 cm and 0.7 cm deep) in the tuber surface with a cork borer. The tubers were inoculated

Phytoalexin

accumulation

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337

immediately with 0 1 ml of bacterial suspensionper trough. For the smaller S. tuberosum ssp.andigenatubers, 0.05 ml of inoculum per wound (0.4 x 0.7 cm and @4 cm deep) was used. In control experiments the potatoes were inoculated with phosphate buffer. The inoculated potatoes were incubated at 15 “C in the dark. They were placed into large desiccators, where a humid atmosphere was maintained by placing moist filter paper below and on top of the tubers. To prevent the tubers from becoming anaerobic, the desiccators were flushed continuously with a stream of air (2 1h-‘) saturated with water. Hole inoculationmethod In a second inoculation method three holes (0.5 cm in diameter and 1.5 cm in depth) were cut into surface-sterilized tubers with a cork borer. The bacterial suspension (2.5 x lo6 cells in 005 ml) was put into each hole, the tuber tissueplugs were replaced and the wounds sealed with Vaseline. Each tuber was wrapped with moist filter paper and incubated at 20 “C in a 500 ml covered glassvessel (Week and Company, Wehr, Germany). The method resembled that described by Lapwood et al. [W] and Lyon [19]. Only the preliminary experiments with cv. Sieglinde were performed using this method. Measurementof amountof rotting After different incubation periods (1-7 days after inoculation), one to four tubers were taken for analysis. Just prior to rot removal, the weight of each tuber was determined and the isolated rotten tissue was weighed. The percentage of rot was taken as the parameter of susceptibility. Rots of one incubation time were combined and stored at -20 “C until analysed. For the smaller S. tuberosumssp andigenatubers, rot (if it occurred), tissueadjacent to the rot (between l-3 mm in depth), and tissuecontaining the wound periderm (within l-3 mm of the wound) were combined for extraction. In control tubers the wound periderm (tissuewithin l-3 mm of the wound) was removed and kept for analysis. Extraction and estimationof phytoalexinr We modified the method of Lyon [19] to extract the tissue (@5-6 g). After stirring for 1 h with ethanol (15 ml g-’ fresh wt) the extract was centrifuged at 5100g for 12 min. The residue was re-extracted once and the combined supernatants were reduced in vacua at 40 “C. The residue was dissolved in 100 ml of a mixture ofwater/ethanol[2 : 3 (v/v) ] and extracted three times with 100 ml dichloromethane, allowing the phases to separate for 1 h. The combined dichloromethane extracts were reduced in vacua to about 80 ml and dried overnight over anhydrous sodium sulphate. The filtered extract was dried completely under reduced pressure and redissolved in 1.5 ml of dichloromethane. We used ethanol instead of the methanol used by Lyon [19], because cislinoleic methyl ester interfered with rishitin diacetate during GC, whereas the ethyl ester of the acids did not. To measure rishitin reproducibly by GC, it was necessary to derivatize the extracts with acetic anhydride/pyridine at room temperature overnight. This method of

338

S. Hildenbrand

and H. Ninnemann

acetylation proved superior to acetylation with acetic anhydridelpyridine at 80 “C for 2 h or treating with trifluoroacetic anhydridelpyridine at room temperature. The latter two methods destroyed someof the phytoalexins. The excessof the reagents was removed by gently blowing nitrogen into the vial, which was kept at 37 “C. The residue was redissolvedin dichloromethane. The maximal variation in parallel extractions was 10%. Recently, we improved the method for e$racting phytoalexins by using the Extrelut” (Merck) column extraction, which is based on liquid/liquid partitioning [II]. Small columns were prepared by dry-packing glass syringes (2 x 10 cm) with 2.5 g Extrelu?. This method was used for probes from 0.5-3 g fresh wt. Each sample was extracted twice with ethanol (15 ml g-’ fresh wt). After centrifugation the combined ethanolic supernatants were dried completely under reduced pressure at 40 “C in round bottomed flasks. The residue after suspending three times in 1 ml of distilled water, was applied to the ExtrelutQ matrix and allowed to stabilize for 30 min. Then the round bottomed flasks were washed three times with 5 ml of l-butylmethyl ether and the solvent used to extract the lipophilic substancesfrom the Extrelut@. The flow rate was reduced to 1 ml min- ’ by fixing a metal cannula on the syringe. The ether fractions were collected, dried completely in VQCUO and redissolved in dichloromethane. The extracts were derivatized and redissolvedin dichloromethane as described above. This method has several important advantages. Firstly, the elution is continuous. Secondly, the recovery yields of the phytoalexins are better compared to the described modified method of Lyon. In addition, the solvent consumption is much less and scarcely any halogenated solvent is used for extraction. Operation time is reduced (30 min fixing time and 30 min elution time) and no drying is necessary.Handling is simple and easy to carry out, allowing more samplesto be extracted at the same time. Gas chromatography

Samples of 2 pl of the derivatized extracts were injected into a glass column (150 mm x 10 mm outer diameter) filled with 100 mg Chromosorb W-AW-DMCS (80-100 mesh) which was held in place with silanized glasswool plugs [13]. Before use, the pre-column was conditioned by heating at 270 “C for 20 min under a helium flow. The volatile compounds of the extract were thermically desorbed (230 “C, 20 min) and transferred by a helium stream in split mode onto a capillary column. The entrance splitting ratio during the transfer was 1: 10. For separation, a capillary column (DB1301,J&W Scientific, 30 m, inner diameter 0.32 mm, thickness of film 0.25 pm) was used in a programmed mode: 10 min at 30 “C, followed by a temperature increase to 280 “C at 5 “C min-‘. The injection port and detector temperature were 230 “C. Helium at a flow-rate of 3 ml min-* at 20 ‘C was used as the carrier gas [12]. For routine measurements, GC (Model 5890, Hewlett-Packard) in combination with a flame ionization detector was used. For identification of the compounds we used GC (Model 5790A, Hewlett-Packard) in combination with electron impact ionization (EI) ms (Model 5970A, Hewlett-Packard). The phytoalexins were identified by comparison with reference samplesof authentic rishitin, solavetivone, phytuberin and lubimin and with spectra in the literature. For quantification, no-decanoic acid methyl ester was used as internal standard.

Phytoalexin

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tubers

RESULTS Constituents of potato soft rot extracts The separation of the constituents (their identity was confirmed by MS) of a typical extract of Erwinia-induced soft rot is shown in Fig. 1. The most prominent phytoalexin was rishitin (diacetate, peak 11); smaller amounts of solavetivone (peak 7) and phytuberin (peak 6) were also measured. Phytuberol (peak 4) and dehydroxyphytuberol (peak 1) [MS m/e (rel. int.) : 234 [M+, 32), 219 (7), 205 (67), 189 (94), 173 (lo), 161 (lo), 133 (23), 107 (48), 93 (55), 81 (62), 67 (41), 55 (49)] were found in very small amounts. Probably they were not genuine but derived from phytuberin during thermal desorption. Therefore, we summed these three phytoalexins. No lubimin was found. In some potato extracts small amounts (10 pg g-’ fresh wt) of hydroxysolavetivone (acetate, peak 15) were detected. Occasionally we detected a substance which, basedon the EI massspectrum (Fig. 2) [characteristic ions m/e (rel. int.) : 2 18 (M+, 22), 190 (71), 120 (87), 105 (56), 91 (59)], was considered to be solanascone.To our knowledge this is the first report of solanascone in potato, although this

r

7

11 16

2

3

4 I.&LLJIL 5

, 1

30 FIG. 1. Typical

I

35

40 Time (mm)

8

I

45

_

50

gas chromatogram of an extract from potato tubers inoculated with Erwinin ssp afrosepica and incubated at 15 “C. The major compounds corresponding to peaks 1-16 were identified by MS as: peak 1, dehydroxyphytuberol; 2, dihydroactinidiolid (standard); 3, 2,4-dichlorophenoxy acetic acid methyl ester (standard) ; 4, phytuberol; 5, myristic acid ethyl ester; 6, phytuberin; 7, solavetivone; 8, palmitoleic acid ethyl ester; 9, palmitic acid ethyl ester; 10, steroisomer of rishitin diacetate; 11, rishitin diacetate; 12, cis-linoleic acid ethyl ester; 13, Qlinolenic acid ethyl ester; 14, stearic acid ethyl ester; 15, hydroxysolavetivone acetate; 16, no-decanoic acid methyl ester (internal standard). cardouora

S. Hildenbrand

340

and H. Ninnemann

. 1400 190 1200 PI ii. n u

1000 000

5 f

600 400

0

40

60

FIG. 2. Electron

00

100 Mass/Charge

impact

ionization

120

140

mass spectrum

160

190

200

of solanascone.

sesquiterpenoidhas been detected in Nic&na spp. [34]. A substance(peak 10) near the rishitin diacetate peak was always observed, and was usually about 4% of the rishitin content. This compound had a massspectrum very similar to that of rishitin diacetate and we consider it to be a steroisomerof rishitin. For the structural formulae of these phytoalexins, the reader is referred to Sato et al. [31] and Uegaki el al. [33,34]. Various fatty acid ethyl estersconsistingmainly of (in decreasingamount) cis-linoleic acid, a-linolenic acid, palmitic acid, palmitoleic acid, stearic acid and myristic acid were also detected. Only palmitic acid was detected as the free acid. Sometimes heneicosaneand the ethyl ester of arachidic and heptadecanoic acid were found. The free fatty acids probably arose from enzymatic breakdown of lipids present in the original plant tissue[26]. They were esterified to the ethyl estersduring extraction with ethanol; when the tissuewas extracted with methanol the corresponding methyl esters were found. Extracting freeze-dried tissue with dichloromethane yielded palmitic acid and no estersof fatty acids. In infected tissueof unstored potatoes which were very susceptibleto E. carotovora sspatroseptica, the concentrations of palmitic, cis-linoleic and a-linolenic acid were higher than in stored tubers, which were lesssusceptible. In control tubers, normally only small amounts of fatty acid ethyl esters could be measured. In tubers inoculated using the hole method, palmitic acid and the methyl esters of palmitoleic and a-linolenic acid were identified after extraction with methanol. In all testedpotato cultivars (cvs Grata, Irmgard, Datura and Hansa) and accessions ofS. tuberosum ssp andigena, the spectrum of compounds was identical and no compound was unique to any genotype. Kinetics of tuber decay and phytoalexin accumulation During infection, the inoculated tissuegenerally changed to soft rot or it produced a thick, dry, grey wound periderm. The control tubers produced a very thin yellowishwhite wound periderm.

341

Phytoalexin accumulation

in potato tubers

FIG 3 Time-course ofrotting ofpotato tubers (cv. &a&a) inoculated with &~~inkI C~~O~OuD~~ SsP a~$& (m). Controls (a), are adjacent to the inoculated probes.

40

r

30 -

r

FIG. 4. Time-course of rotting of potato tubers (cv. Irmgard) inoculated with Envinin ssp a~rusep~ica[m), Controls (Q), are adjacent to the inoculated probes.

~nrol~uor~

S. Hildenbrand

and H. Ninnemann

FIG. 5. Time-course of solavetivone (m), rishitin (a) and phytuberin (sum of phytuberin, in tubers of cv. Grata infected phytuberol and dehydroxyphytuberol) (m) accumulation with Ertuinia carohmra ssp ahwptica. Controls (Cl), are adjacent to the inoculated probes.

FIG. 6. Time-course of solavetivone (IJJll), rishitin (0) and phytuberin (sum of phytuberin, phytuberol and dehydroxyphytuberol) (W) accumulation in tubers of cv. Irmgard infected with Erzuinia carohora sspalrosep!ica. Controls (a), are adjacent to the inoculated probes.

The difference in susceptibility of stored tubers of the cvs Irmgard and Grata was not large enough to get clear differences in rotting and phytoalexin response. The collection of German cultivars has many genotypes with intermediate or high susceptibility but no resistant cultivars [5,9,37]. Stored tubers of cv. Grata rotted more slowly at the beginning than cv. Irmgard, but from the third day on they rotted more easily than cv. Irmgard (Figs 3 and 4). Rotting in both cultivars was lessthan 10 o/oin the first 6 days. In tubers of cv. Grata, rishitin accumulated rapidly after 24 h and

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tubers

reached a maximum of 500 l.tg g-l fresh wt 3 days after inoculation. Solavetivone and phytuberin accumulated in smaller amounts, with maxima of 180 and 150 l.tg g-l fresh wt respectively, at 5 days after inoculation (Fig. 5). In tubers of cv. Irmgard, rishitin accumulated to a similar maximum, but less rapidly (Fig. 6). Solavetivone and phytuberin accumulated in very small amounts with maxima at 5 days after inoculation of 20 and 40 l.tg g-’ fresh wt, respectively. Using immature tubers, which were inoculated 1 day after harvesting, both cultivars rotted very fast and reached an extremely high percentage of rotting (Figs 3 and 4), with cv. Grata being more susceptible than cv. Irmgard. In both cultivars no phytoalexins were detected in the infected rotted tissue. All control tubers wounded using the trough method accumulated, at best, traces of phytoalexins in the tissue containing the wound periderm. This is in agreement with the experience that wounding [29] and buffer components (G. D. Lyon, pers. comm.) can elicit stressmetabolites. In intact, healthy potato tissue no phytoalexins were measured. When tubers of cv. Sieglinde were inoculated using the hole method, rotting was extensive. After 6 days, 30% of the tissue was creamy, watery and foul smelling; the controls did not rot. No phytoalexins were measured in the extracted rotted tissues. The conditions at the rot site were probably anaerobic and Clostridium sspand/or other indigenous microorganisms were probably involved in rotting [17,18]. In contrast, the rotted tissueofstored and unstored tubers inoculated with E. carotovora sspatroseptica using the trough method were never foul smelling (see Stapp [32]). Tubers of S. tuberosum ssp andigena were scarcely susceptible. Rotting in both accessionswas lessthan 2 o/oin the first 3 days; often no rot occurred (Table 1). At the

TABLE

1

Susccptibili~ of, and phytoalexin concentration in, luber tissue [combination of rot, tissue adjacent to rot (within 3 mm) and woundperiderm tissue (wifhin 3 mm)] of two accessions of Solarium tuberosum ssp andigena inoculated wifh Erwinia carotovora ssp atroseptica Solarium tuberosum ssp andigeno CIP-No.

703667

Controlt CIP-No.

Controlt

702299

Time after inoculation (h)

Rotten tissue (%)

22.7 47.9 72.7 95-o

0.3 ND 1.3 0.0

194 3853 573.8 126.5

1.2 0.6 2.3 1.4

62 13.2 190 10.6

72.7

0.0

10.6

o-0:

190

198 45.1 699 ‘1160

91 0.0 0.0 12.5

1404 231.7 8958 403.0

8.3 105 633 234

70.0

0.0

0.0s 1.6 260 47 0.01

*Sum of phytuberin, phytuberol tContro1 tuber inoculated with fLess than @5 pg g-i fresh wt. ND, Not determined. 23

Rishitin

Solavetivone

Phytuberin*

(pg g-r fresh wt)

0.01

and dehydroxyphytuberol buffer only.

8.6

concentrations.

MPP44

S. Hildenbrand and H. Ninnemann 344 sametime high concentrations of phytoalexins accumulated, with rishitin as the main sesquiterpene. Accession 702299 accumulated rishitin faster than accession 703667. The extracted tissueconsistedof a combination of rot (if it occurred), tissueadjacent to rot (between l-3 mm) and a defensive wound periderm (between l-3 mm). The actual concentrations of the phytoalexins in the rotted tissuemay be even higher than the values given here for the combined tissues,becauseadditional experiments showed that phytoalexins were located mainly in the rotted tissue.

DISCUSSION

The aim of our experiments was to investigate the possibleparticipation of phytoalexins in resistance of potato tubers against E. carotovora.Little information exists in the literature about the speedat which phytoalexins can be induced by Erwinia spp. [Zl]. In the rotted tissuesproduced during the shortest incubation time (20 and 24 h), we detected high concentrations ofrishitin. Rijber [27] measuredlubimin accumulation at 23 h and rishitin and other phytoalexins about 40 h after inoculation of tuber halves with E. carotovorasspatroseptica.In the potato tuber/Phytophthora interaction, 20 h after slicing and immediate inoculation with an incompatible race of P. infestans,rishitin (< 10 pg g-’ fresh wt) was detected [30], whereas rishitin biosynthesis (measured as incorporation of acetate-2-14Cinto rishitin) had started after 30 min in response to slicing tubers [29]. The maximum amounts of rishitin were normally obtained between 3-5 days after inoculation. These concentrations were in the range found to be toxic to E. carotovora sspatrosepticuin vitro [22]. Rishitin, solavetivone and phytuberin concentrations were comparable with those measured by others working with Erwiniu spp [2,19]. The concentrations of rishitin were much higher than in potato tuber tissue infected with P. infestans[IO, 281: this was perhaps becauseof variation in experimental methods, in tuber storage periods [19], the ageing of the tuber slices in investigations with Phytophthora,differences in the pathogens or diseasedevelopment. Lubimin was never detected in our experiments. This phytoalexin was reported to be produced only at very low concentrations in the potato/Erwiniu interaction [21], whereas in potato/ Phytophthoru interactions its concentration was substantial [IO, 211. Perhaps the conditions produced by the pathogens’ attacks were different (e.g. pH; G. D. Lyon, pers. comm.), or different pathogens elicit different phytoalexins [8]. In addition to the phytoalexins, the most conspicuousgroup of substancesfound in the rotted tissuewere the ethyl estersof various fatty acids. High concentrations were detected in rotted tissue,whereas in the wounded control tissuelower concentrations were present. The breakdown of plant cell membranesinduced by pathogens may lead to high concentrations of free fatty acids which can causeanalytical problems [2S]. In our systemwe could separate and measurephytoalexins and most of the fatty acids as ethyl esterswithout interference problems. Somefatty acids have elicitor activity, while others are activated in the presenceof glucans isolated from P. infestuns[25]. Recently, Cohen et al. [3] showed that arachidonic and eicosapentaenoic acids, linolenic and linoleic acids, were efficient elicitors of systemic-induced resistanceagainst P. infestam. It is believed that plants can releaselinolenic acid after wounding or in pathogenesis, leading to synthesis of jasmonic acid [7], a signalling component of defensive and

Phytoalexin

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345

tubers

developmental reactions [35] which might also participate in the horizontal resistance of plants. In the potato/Ertuiniu interaction the production of phytoalexins probably contributes-among other mechanisms-to tuber resistance [,?I]. Our results point in the samedirection: they show that the expression of resistance and the concentration of phytoalexins can be affected by the chosen resistance test (inoculation/incubation method) and physiological age of the tubers. Using the hole inoculation method, tubers rotted to a high degree and phytoalexins could not be detected. Hole inoculation probably leads to anaerobic conditions, under which phytoalexin biosynthesis may be reduced [Xl. Our experiments showed that under anaerobic conditions tuber susceptibility increased, which agrees with reports in the literature [4,18]. Using an inoculation method where the tubers were wounded by making shallow troughs, stored mature tubers showed reduced susceptibility and high concentrations of phytoalexins were accumulated in the rotted tissue.In marked contrast to mature tubers, immature unstored tubers inoculated with this method rotted extensively and no phytoalexins were detected. The immature control tubers produced small amounts of phytoalexins, showing that this tissuehad the ability to synthesize phytoalexins. R6ber [27] found only traces of phytoalexins in mature tubers inoculated immediately after harvest with E. carotovorasspatroseptica. Many different methods of testing potato tuber resistanceto Erwinina spp have been published in the literature [36]. The development of soft rot is easily influenced by various parameters of different tests. This could account for published contradictory results on the influence of storage length on tuber soft rot susceptibility [4, II, 16,241. In order to compare different genotypes in a single experiment the tubers must have the same history, i.e., they should have been grown in the same field, been harvested on the same day and stored identically. Variation in soft rot resistance may be partly due to differences in the speed of phytoalexin accumulation or the concentration attained, as was shown in compatible and incompatible potato/Z’hytophthoru interactions [28,38], as well as in compatible and incompatible cotton/Xanthomonas [6] and in other interactions [I]. Our experiments with the German cultivars showed a correlation between a very high susceptibility of the anaerobically inoculated tubers or the infected immature unstored tubers and the lack of phytoalexins. Stored tubers of cv. Grata tended to produce phytoalexins more rapidly than tubers of cv. Irmgard, although the differences in susceptibility were not unequivocal. The selected accessionsof S. tubermum isp an&gena however, showed very distinctly the correlation of high concentrations ofphyto&&s with low susceptibility. Experiments with susceptible German cultivars in comparison with selected lesssusceptible genotypes from the International Potato Center in Peru are being evaluated at present. We wish to thank L. Schilde-Rentschler for valuable discussionsand helpful advice; F. Jiittner (Ziirich) and K. Wurster for assistancewith GC-MS. We thank H. R. Hohl (Ztirich) for supplying samplesof solavetivone, phytuberin and lubimin, G. D. Lyon (Dundee) for a sample of phytuberin and A. Murai (Sapporo) for samplesof rishitin, solavetivone and lubimin. 23.2

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S. Hildenbrand

and H. Ninnemann

.

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