J. Phytopathology 117, 97—106 (1986)© 1986 Paul Parey Scientific Publishers, Berlin and HamburgISSN 0031-9481

Institute of Potato Research Grojl Liisewitz of theAcademy of Agricultural Sciences of tbe GDR

Virulence and Enzyme Production ofErwinia carotovora ssp. atroseptica on Potato Tuber Tissue

J. WEBER and CHRISTINA WEGENER

Authors' address: Dr. JURGEN WEBER and Dr. GHRISTINA WEGENER, Institute of Potato Research ofthe Academy of Agricultural Sciences of the GDR, 2551 Grofi Liisewitz (GDR).

With 4 figures

Received October 30, 1985; accepted May 30, 1986

Abstract

The elicitation of pathogenesis on potato tuber slices by 6 strains of Erwinia carotovora ssp.atroseptica was investigated by neutral red vital staining and has been compared with bacterial growthrate, penetration ability, enzyme production and enzyme spectrum. The induction of enzymesynthesis (particularly, of the extracellular polygalacturonase) elicites the rot attack on tuber tissue andthis requires a sufficient bacterial density.

Due to wound healing, the inductors of enzyme production are removed, and after 48 henzymes do not attack tuber tissue any more. Therefore, growth rate and penetration ability (to getthe necessary bacterial density and inductor substances) may limit virulence. A similar influence ofenzyme production and enzyme spectrum of the strains on the virulence was not detected.

Zusammenfassung

Virulenz und Enzymproduktionvon Erwinia carotovora ssp. atroseptica auf KartoffelknoUengewebe

Die Auslosung der Pathogenese auf KartoffelknoUenscheiben wurde iiber eine Neutralrot-Vitalfarbung an 6 Stammen von Erwinia carotovora ssp. atroseptica untersucht und mit derenWachstumsrate, Penetrationsvermogen, Enzymbildungsvermogen und -spektrum verglichen. DieInduktion der Enzymproduktion (speziell extrazellulare Polygalakturonase) leitet den Gewebebefallein, wozu eine ausreichende Bakteriendichte erforderlich ist. Infolge der Wundheilung werden dieInduktoren der Enzymsynthese entzogen und nach 48 h wird das KnoUengewebe durch Enzymenicht mehr angegriffen.

Wachstumsrate und Penetrationsvermogen (Erreichen der erforderlichen Bakteriendichte undInduktoren) konnen daher die Virulenz limitieren. Ein Einflufi von Enzymbildungsvermogen und-spektrum der Stamme wurde nicht gefunden.

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98 WEBER and WEGENER

Rot diseases are based on enzymatic degradation of tissue. Consequently, ahigh productivity of extracellular enzymes is considered a general principle ofpathogenicity of rot causing bacteria (MUSSEL and STRAND 1976). Erwinia caroto-vora ssp. atroseptica (= Eca) is known to secrete several enzymes such aspectatelyase (PL), Cx-cellulase, proteinase, polygalacturonase (PC), andphosphatidase. Because of the high secretion rate, pectatelyase is regarded themost promising enzyme for inducing and governing tissue maceration (ZUCKER

and HANKIN 1970, TSENG and MOUNT 1974). The enzyme production of Erwiniais induced by oligomere galacturonides derived from the plant cell wall pectin(CoLLMER and BATEMAN 1982).

In the work reported here, the bacterial density needed for rot attack wasestimated as a criterion of virulence. These data were compared with the enzymeproducibility and the growth rate of bacteria, the ability to penetrate and othercharacteristics studying the variation in virulence of different Eca strains. Theapplication of a sensitive maceration test on potato tuber slices based on neutralred vital staining allowed a detailed investigation of the initial phase ofpathogenesis.

Materials and Methods

1. Organism and culture conditions

Six wild type strains of Erwinia carotovora ssp. atroseptica selected from the culture collectionof the Institute of Potato Research Grofi Liisewitz and six subisolates, derived from the strain G17 byplating bacteria on selective agar medium (Gallensalz-Lactose-Agar from GERMED, Immunprapa-rate Berlin), were used in these experiments.

For estimating the growth rate (by measuring the turbidity against a numbered standard) thestrains were cultured for 24 h at 20 °G on a reciprocal shaker in Erlenmeyer flasks containing 20 ml ofpectin medium, pH 7.0, after ZuCKER and HANKIN (1970).

For the estimation of enzyme producibility a mineral salt medium, pH 7.0, after ZuCKER andHANKIN (1970) with 20% potato cell sap and 15 g/1 potato fibres (= potato medium) or with 10 g/1glucose as single carbon source (= glucose medium) were used.

2, Enzyme assays

Gultures were centrifuged at 15,000 g for 15 min. 1 ml of an appropriate dilution of culturefiltrate was incubated for 15 min to determine the

— Pectatelyase activity after ALBERSHEIM et al. (1960) with 10 ml of 0.1 % polygalacturonic acid in0.1 M Tris-HGl buffer, pH 8.0, (+ 10^ M GaGli) at 25°G. The optical density was measured at235 nm.1 Unit = 1 ;UM unsaturated galacturonides • ml""' • min""'

— Polygalacturonase activity with 5 ml of a 0.8% solution of citrus pectin in 0.1 M Me Ilvainebuffer, pH 4.5, at 30°G. The decrease in viscosity (expressed in %) was measured in an Ostwaldviscosimeter.

— Gellulase activity with 5 ml of 0.6% Garboxymethylcellulose solution in 0.1 M phosphatebuffer, pH 5.1, at 37°G. Decrease in viscosity (%) was measured.

— Protease activity according to DOUNG VAN QUA et al. (1981) with 3 ml of 1 % casein solution in0.1 M Tris-HGl buffer, pH 8.0, at 37°G. The reaction was terminated by the addition of 3 mlof 10% trichloracetic acid. The released tyrosin was determined in the supernatant with 1 ml of1 % a-Nitroso-P-Naphthol in 0.1 N NaOH by measuring the optimal density at 510 nm.1 Unit = 1 /iM Tyrosin • ml"' • min"'

— Phosphatidase activity was determined using the "cup plate" assay (TsENG and BATEMAN 1968).The corona — development in 1 % soybean lecithin — agar medium, pH 8.0, was estimated.

Virulence and Enzyme Production of Frwinia carotovora ssp. atroseptica 99

3. Maceration test

Twenty slices of medullary tuber tissue from cultivar 'Adretta' (10 mm in diameter, 2 mmthick) were incubated for 16 h at 20°C in petri dishes between two filter papers dipped before in Fca-suspension, enzyme solution or water (for wound healing or control). After incubation, the sliceswere shaken in water for 1 h and stained with neutral red (50 ppm neutral red in 0.2 M phosphatebuffer, pH 7.5, with 100 g KNO3 per liter) for 90 min. The rinsed slices were treated twice with 10 mlof 96 % ethanol for 15 min to extract the absorbed neutral red and the extracts were filled up to 50 mlwith 0.02 N H2SO4. Tissue maceration (% degradation) was estimated by measuring the opticaldensity at 535 nm against a water-treated control (= 100% vital staining).

4. Penetration ability

Sterile FN4-paper strips were inoculated in punctuate way with 0.05 ml containing 5 x 1 0 ^ Fcacells (Fig. la) and dipped for 16 h in 1 : 5 diluted pectin medium in 0.1 M citric acid/phosphatebuffer, pH 5.8. After the flowing period the serrated parts of paper strips were stamped (Fig. 1 b) onselective agar medium dishes (method 1) and incubated for 2 days at 28 °C. The migration distance ofthe bacteria was estimated with ± 1 cm reproducibility by counting off the right/left 5 mm displacedpunctures and/or fc^^-colonies (Fig. 1 c).

5. Extraction of enzymes from potato tuber tissue

150 tissue slices were incubated with Fca for several hours (method 3). The water rinsed shceswere homogenized for 1 min (BUHLER-Homogenizer) in 50 ml 0.1 N NaCl-solution and centrifu-gated for 10 min at 15,000 g.

Fig. 1. Determination ofbacterial migration throughcapillary tubes of FN4-paperstrips (a) by counting off thepunctures and/or developedcolonies (c) after stamping on

selective medium (b)

100 WEBER and WEGENER

6. Detection of galacturonides

150 tissue slices (fresh or after 24 h of wound healing — method 3) were rinsed twice with 50 mlof water, the solution was vacuum evaporated and the residue resolved in 10 ml of 80 % ethanol. Aftera short boiling macromoleculare substances were removed by centrifugation (15,000 g, 20 min). Thetotal galacturonide content was determined with thiobarbituric acid (NEUKOM 1960) and the galac-turonides were identified by paper chromatography (NAGEL and VAUGHN 1961).

7. Enzymes used in the experiments

Pectatelyase (of Erwinia carotovora) was precipitated from culture filtrate by 60 % (NH4)2SO4,dialysed, partially purified by gel chromatography (FRAGTOGEL TSK HW-55 [F], MERGKDarmstadt) and lyophilized.

Pectinmethylesterase (of oranges) was prepared after KEIJBETS (1974).Gellulase (of Penidllium) we obtained from Humboldt University Berlin, department of

Microbiology, pectinase (of Aspergillus) and proteinase (of Bacillus subtilis) from SERVA, Heidel-berg.

Results

Vital staining with neutral red is very sensitive, and the measurement oftissue degradation is reproducible at a high rate (s % = 5 using uniform tuber andinfection material).

Changes in the tissue's texture become externally visible only beyond 30 %of maceration. With tissue degradation ranging from 15 to 80% the degree ofmaceration revealed linear dependence on the logarithmus of inoculum density(Fig. 2 a). In this range the inoculum density was found to determine only thedegree of maceration. A bacterial density causing a maceration of 20% within16 h at 20°C was able to overcome defense reactions of the tissue and to causeprogressive rot. This inoculum density (= log Ecai) of bacterial strains wasdetermined by diagram (Fig. 2 a) and was considered to characterise the virulenceon fresh potato tissue. After a wound healding period of 24 h at 20 °C, theinoculum density necessary to cause a maceration of 20% was found nearly 100

48 hw

8 9 0 1 2 3

Erwinia (log. Ecamt" ' ) pectatelyase (IgmUmI" ')

Fig. 2. Maceration of tuber tissue (fresh cut slices (f) or after wound healing period (w)) in relation toinoculum density of Erwinia (a — strain G 17) and pectatelyase activity (b)

Virulence and Enzyme Production of Erwinia carotovora ssp. atroseptica 101

Table 1Characteristics of virulence of some Eca strains on potato tuber tissue (log Eca{ = logarithmic value ofmaceration inducing Eca density on fresh tissue, log Eca^ = inducing density after 24-hour wound

healing, variation of 3 X repeated cultures)

Strain

C17CP12111103CP71P63

log Ecai

6.0—6.46.0—^.25.9—6.56.4—^.66.0—6.26.6—6.8

(Range)

111213

log Eca^

8.2—8.78.6—8.88.8—>98.6—9.0>9>9

(Range)

123244

(2 range)

234457

times higher due to wound healing reaction (log Eca^ as an additional marker ofvirulence — Fig. 2 a).

The degree of virulence of strains differed due to the log Ecar and log Eca^-values (Table 1). However, no correlation occurred between virulence andenzyme producibility of strains. Maceration tests with pectatelyase showed, thatonly 30 mU of pectatelyase on fresh tissue or 100 mU of pectatelyase after 24 h ofwound healing are needed to provoke 20 % tissue maceration (Fig. 2 b). Thesevalues were exceeded by culture filtrates of all Eca strains, regardless of the widerange of variation (Table 2).

Also the spectrum of enzymes did not determine the graduation of virulenceof the strains (Tables 1 and 2), although mixtures of enzymes undoubtedly speedup maceration (Fig. 3).

Pectinmethylesterase (produced by Eca in very small amounts, but presentin potato tissue) and protease had no macerating effect on tuber tissue, butcombined with pectatelyase they increased the effect of this enzyme markedly.

The addition of polygalacturonase and cellulase also increased maceration.The start of tissue maceration and enzyme production depends on the

inoculum density (Table 3). In the case of low densities several hours are neededfor bacterial multiplication to start maceration. The tissue decay performed by an

Table 2Enzyme production of some Eca strains in "potato medium" (variation of 3 X repeated cultures,

GM = glucose medium)

Strain

C 17CP12111103CP71P63

Pectatelyase

(U • ml-')

2.9— 8.55.4— 7.83.3— 9.67.0—10.27.4—12.74.2—12.8

inGM(U • 10-'Eca cells)

0.150.110.170.140.250.25

Polygalact-uronase

C^-Cellulase

(% viscosity decrease)

5.8—14.55.6— 9.2

10.5—24.811.8—16.35.4—15.95.6—10.6

32.9—49.525.3—37.613.5—27.329.8—33.636.9—39.842.J—46.5

Protease

(U • ml-')

0.8—1.80.4—1.00.1—0.70.40.1—0.8

. 0.4—0.8

Phosphatidase

(mm corona)

3.01.00.502.02.0

102 WEBER and WEGENER

Table 3Tissue maceration and enzyme production of Eca strain C 17 in relation to incubation period andinoculum density (a = 10*, b = 10*, c = 10', d = lO* Eca • ml"', enzyme activity of extracts of

inoculated tuber slices)

Incub.period(h)

048

16244048

After

a

019297390——

4.3

maceration

b c d

01239566973

9

0215768

22

02326

39

PG-activity(% vise, decrease)

a b e d

0

03036 036 3 0 0— 13 7 4

h = 20 % maceration.

a

0

00.10.51.2

PL-activity(U • ml-̂ )

b e d

0 00.1 0.1 00.8 0.6 0.3

a

6.0

6.87.7—8.9

pH-value

b c d

6.77.5 7.3 6.7

inoculum of 10"* Eca cells • ml * after 39 h was stopped by defense reactions of thehost.

Results of Table 4 point to a relationship between the growth rate of strainsand the virulence.

In the case of high bacterial density only a short period for attacking tubertissue is required.

The induction periods of strains (= period between inoculation with 10̂ Ecacells ml~̂ and start to attack defined by 20% maceration) were ascertained bymeasuring the course of maceration. Owing to the small variation there is norelationship to the virulence of strains (Table 4).

The biosynthesis of polygalacturonase (PG) started earlier than the produc-tion of pectatelyase (PL) (Table 3), which occurred only when the pH valueshifted towards the alkaline range.

The PG (pH optima — 5.0) enables Eca to macerate tuber tissue at pH 3.0,whereas PL with an optimum at pH 8.0 started a measurable maceration at pH6.0 (Fig. 4).

co

uo

60-1

2 40-

20-

effect without PL (ca. 500mUmr')

additional effect with PL

+ PME PG cellulase protease PL (addition)

Fig. 3. Maceration activity of pure and with pectatelyase combined enzymes on potato tuber tissue(PME = pectinmethylesterase, enzymes solved in water)

Virulence and Enzyme Production of Erwinia carotovora ssp. atroseptica 103

Table 4Maceration inducing period (a), bacterial growth rate (in pectin mediiun = b) and penetration abiUty

(c, variation of 3 X repeated cultures) of some Eca strains

Strain

G 17GP 12111103GP71P63

ah t o 2 0 %maceration

4.05.04.63.65.45.0

bBact. density

ml-* after 24 h

6.6 X 10' cells5.76.56.45.64.9

(Range)

121123

ccm migrationon FN4 paper

13—1712—155—105—103—63—6

(Range)

123344

(2 range)

244467

On the wound surface of fresh cut tuber slices (cultivar 'Adretta') 4.2 mggalacturonides (total amount per cm^ wound surface) were found, as determinedin the water after rinsing the slices. Galacturonides were identified by paperchromatography. The galacturonic acid, which does not induce the bacterialenzyme production, formed the main part of the galacturonides (ca. 70%).Active inductor substances (dimere and trimere galacturonides) were detectableonly in traces. After a wound healing period of 24 h the total amount ofgalacturonides decreased to 0.4 mg • cm~̂ of wound surface (Table 6).

When inoculations were carried out after a wound healing period of 24 h,tissue maceration was only caused by an inoculum of > 10̂ Eca cells ml~̂ (Fig-2 a, Table 1). After a period of 48 h maceration was also not performed by PL(Fig. 2 b). Thus the possibilities of pathogenic attack are restricted by woundhealing processes.

The basal PL activities, as estimated after culturing the strains in glucosemedium (repressing inductive enzyme synthesis — HUBBARD et al. 1978) did notinfluence the virulence of the strains (Tables 1 and 2). Basal PG activities have notbeen found.

oSuoE

80-

60-

40-

20-

2 3 4 5 6 7 8 pH-value

Fig. 4. pH-dependence of maceration activity of Erwinia (strain C 17) and pectatelyase on potatotuber tissue (Teorell Stenhagen buffer used)

104 WEBER and WEGENER

Table 5Variation of characteristics (see Table 1 . . . 3) of some subisolates of the strain C 17 (data of virulence

and penetration from 3 X repeated cultures)

Isolate

123456

Virulencelog Ecai

5.9—6.06.0--6.26.0—6.26.0—6.26.0—6.25.9—6.8

log Eca^

8.3—8.88.3—8.58.7—9.08.7—8.88.7—>98.7—>9

PG(% vise. deer.

9.75.08.17.66.38.4

PL) (U • ml-')

4.46.55.75.54.53.1

Growth rate(X 107ml/cells)

7.26.66.56.15.84.5

Penet. ability(cm migration)

18—191̂ —1913—1713—1712—1512—15

Owing to their mobility the £ai-bacteria are able to penetrate into tubertissue. This ability has been characterised by migration distances of Eca cellsthrough capillary tubes of paper in a medium similar to plant tissue sap (Fig. 1).The migration distances of the strains (Table 4) point to a relationship to thevirulence. Also the data of Table 5 reflect, that the growth rate and penetrationability influence the virulence of the C17-subisolates in a similar way.

Discussion

Erwinia realizes the rot attack by the production of extracellular enzymes.The pectin degradation is started by PG (Table 3). This shows the adaptation ofEca to the acid reaction of healthy tissue. Continued tissue maceration by PL, themain enzyme protein, is an evidence for the adaptation to the alkaline reactionshift of rotting processes.

Owing to the wound healing, it is only possible to start a continuousenzymatic tissue maceration within 48 h after wounding (Fig. 2 b). In this periodthe enzyme production of Erwinia has to be induced and this requires anadequate high bacterial density (Fig. 2 a) and inductor substances derived fromthe host pectin (COLLMER and BATEMAN 1982). Due to the removal of inductorswithin a wound healing period of 24 h, an inoculum of 5 • 10^ Eca cells ml~̂ didnot cause tissue maceration (Table 6). The addition of oligomere galacturonidesto this inoculum triggered off the maceration immediately after the 24 h period.

It is supposed, that the induction of enzyme synthesis by oligomere galac-turonides (liberated from host cell wall by mechanical damages) plays a dominantrole in the host/pathogen relation of ^otzio/Erwinia.

Table 6Effect of galacturonides on tuber tissue maceration (inoculum — 5 10̂ Eca cells • ml"')

Tissue slices Free galacturonides Tissue maceration(mg • cm-̂ slice) (%)

Fresh 4.2 40After 24 h 0.4 0After 24 h + dipped in 0.2 % galacturonide solution 50

Virulence and Enzyme Production of Erwinia carotovora ssp. atroseptica 105

A high inoculum density (> 10^ Eca cells ml"*) is able to attack the tissueafter a wound healing period of 24 h without the addition of inductors. Thehistological findings of Fox et al. (1971) suggest the penetration ability of bacteriato be a factor of virulence: Eca cells pass through spaces of incomplete woundhealing and penetrate into deeper tissue regions, where the enzyme production iselicited by inductor substances present in this region.

The influence of penetration ability on the virulence of bacteria (Tables 4 and5) is comparable in a way with the pathogenicity of rot causing fungi {Fusariumspp.). In spite of a small enzyme producibility they realize rot attack by a highpenetration ability of their hyphes (O'BRIEN and LEACH 1983).

Only small enzyme activities are necessary to elicit rot attack within 24 hafter wounding (Fig. 2 b), therefore the bacterial virulence is determined in firstline by the factors influencing the start of enzyme production: growth rate (forhigh bacterial density) and penetration ability (to overcome the inductor removalby wound healing) (Tables 1 and 3). The start of tissue maceration (this means acompatible host/pathogen relation) is not influenced by the enzyme producibilityof the strains (Tables 1 and 2). This observation is in agreement with the results ofMAHMOND et al. (1981) and TSUCHIYA et al. (1983), but disagrees with the resultsof BuLNHEiM (1978). These different results may be caused by the differentcharacterisation of the bacterial virulence. Characterising the virulence by the rotcausing inoculum density, the enzyme activity (producibility) does not correlatewith this mark, but if the virulence is characterized by the spread of rot (afterincubation with 10^ Eca cells ml"^) a correlation occures (after BULNHEIM 1978, r= 0.922). The spread of rot by enzymatic tissue maceration is linear dependent onPL activity in a range up to 1,000 mU (Fig. 2 b), therefore in this way ofcharacterisation the enzyme producibility determines the virulence.

The differentiation of the strain C17 into 6 subisolates (Table 5) gives animpression of the wide variation of isolates in their quantitative characterisation.Therefore it is difficult to investigate quantitative relationships of virulence.

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