Susceptibility of carrot cultivars to Mycocentrospora acerina and the structure of cell wall polysaccharides

Physiological

and Molecular

Plant Pathology

( 1994)

45, 139-I

139

51

Susceptibility of carrot cultivars to Mycocentrospora acerina and the structure of cell wall polysaccharides B. LE CAM*:,

P. MASSIOTt, C. CAMPIoNj’ and F.

ROUXEL*

* S!alion de Pathologic Vigltale and t Station de Recherches Cidricoles, Biotransformalion Inslilul National de la Recherche Agronomique, B.P. 29, 356.0 Le Rheu, France (Accepted for publicalion

des Fruits

et Ltgumes,

June I994 )

The role of cell wall polysaccharides in pre-formed resistance to necrotrophic fungi is poorly understood. The relationships between cell wall polysaccharides, especially the middle lamella pectin, of four carrot cultivars and their susceptibility to Mycocenfrosporu acerina, a cold storage pathogen, were investigated. Amounts ofcell wall material and composition of the pectic fraction (galacturonic acid, neutral sugars) were not correlated with cultivar resistance. However, amounts of acid-insoluble pectin (protopectin) increased with the resistance of the cultivar. Maceration in uiuo of carrot tissues from different cultivars by M. acerina enzyme preparations, mainly polygalacturonases, correlated with their susceptibility to the pathogen. In the same way, the solubilization rate in uilro by M. acerina enzymes of pectic material isolated from the most susceptible cultivar was I.8 times higher than from the least susceptible cultivar. Hydrolysis of purified middle lamella pectin by endopolygalacturonase confirmed this correlation. Although the degrees of methoxylation of carrot cultivar pectins were similar, their behaviour towards endopolygalacturonase was different, indicating a variation in the distribution of the methoxyl groups along the pectin chain. The results showed a similar composition ofcell wall polysaccharides in the different cultivars but the solubility and esterification characteristics ofpectins suggest that pectic substances have an important role in constitutive resistance of carrot to M. acerina.

INTRODUCTION acerina Hartig Deighton, responsible for liquorice rot, is probably the most prevalent pathogen occurring on carrots in long-term cold storage [8,15]. Observations in planta showed that, after inoculation with chlamydospores whose germination is furthered by wounds, the mycelium progresses from one host cell to the other, leading to the degradation of the intercellular matrix [7]. It is now well established that cell wall polysaccharide-degrading enzymes secreted by pathogens, notably pectinases, are implicated in a process referred to as tissue maceration, by hydrolysing the rhamnogalacturonan present in the middle lamella [10 1. In a previous paper [14], we highlighted that M. acerina secreted ckll wall polyiaccharide-degrading Mycocentrospora

$To whom correspondence should be addressed. Abbreviations used in text: AIS, alcohol-insoluble solid; CDTA, cyclohexane-diamino-tetracetic-acid; CSP, cyclohexan-diamino-tetracetic acid soluble pectin; DM, degrees of methoxylation; endoPG, endopolygalacturonase; HPGPC, high performance gel-permeation chromatrography; HPIEC, high performance ion-exchange chromatography; HSP, HCL soluble pectin; Mv; viscosity-average molecular weights; PG, polygalacturonase; PME, pectin methylesterase; Ve, elution volumes. 0885-5765/94/080

139 + 13 808.00/O

@ 1994 Academic

Press Limited IO-2

B. le Cam, P. Massiot,

140

C. Campion

and F. Rouxel

enzymes in vitro, the production of which was positively correlated with the aggressiveness of the isolate. Challenged with pathogens, plants display a large number .of active defence responses including the synthesis of phytoalexins and the deposition of lignin and hydroxyproline rich glycoprotein in the cell wall, thus leading to lesion limitation [6, II]. In the same way, the polysaccharide structure of cell walls may hinder necrotrophic disease development. Very few studies deal with the role of host cell wall polysaccharides in susceptibility to pathogens and such a pre-formed resistance mechanism is poorly understood. However, relatibnships have been found between the degree of methoxylation of the potato tissue cell wall pectin and susceptibility to Erwinia carotovora [28], as well as between the solubility of strawberries [13] and potato clone pectins [35] and their susceptibility to Botrytis cinerea and Erwinia carotovora, respectively. No carrot cultivar resistant to M. acerina has been reported, but observations of soft rots suggest that cultivars differ in their degree of susceptibility to M. acerina.

This study aimed to investigate correlations between susceptibility to M. acerina and the composition and structure of carrot cell wall polysaccharides, especially the middle lamella pectins. This paper also reports the relationships between in vitro enzymatic hydrolysis of carrot cell wall and pectins, and susceptibility of four cultivars to M. acerina. MATERIALS

AND

METHODS

Carrot roots and fungus

Four carrot cultivars, Major, Flakkee, Boltex and Touchon, were grown in polders of The Mont Saint Michel area (France) at the same site and under identical conditions without fungicide treatment, and were harvested 7 months after sowing and stored at 4 ‘C. The roots were washed with tap water before experiments. Isolates of M. ace&a obtained from typical lesions on carrot roots were grown on malt agar medium and their pathogenicity was maintained by frequent subculture on carrot agar. Two isolates, aggressive (28) and hypoaggressive (30) were selected [14]. Susceptibility

assessment

The susceptibility of the cultivars to M. acerina was evaluated on the whole root by quantifying rot development 5 days after inoculation with a mycelium plug of an aggressive isolate, according to the technique of Montfort & Rouxel [21] and adapted by Le Cam, Massiot & Rouxel [14]. The disease index is the mean of 40 necrosis area measurements. Cell wall polysaccharide-degrading enzymes from M. acerina M. acerina was cultured in a liquid medium without shaking, as described by Le Cam et al. [14], with carrot cell walls as an induction factor. After 7 days growth, cultures

were filtered under vacuum through G3 sintered-glass and then under pressure through 3 pm and @45 pm Sartorius filters. Filtrates were extensively dialysed (10000 mol.wt cutoff) against deionized water at 4 “C and freeze-dried. The lyophilized powder was resuspended in 20 mM citrate phosphate buffer pH 4.5, and the preparations were standardized for their polygalacturonase (PG)’ activity.

Carrot

cell wall polysaccharides

and susceptibility

to M. acerina

141

Pectic enzymepreparation Endopolygalacturonase (endoPG) from Aspergillw niger, purified from Pectinex AR (Novo Industri, Denmark), was provided by the Laboratoire de Biochimie et Technologie des Glucides (INRA, Nantes, France). The endoPG preparation containing 40 nkat ml-’ was in 20 mM citrate phosphate buffer pH 4.5. The pectin methylesterase (PME) preparation from A. niger was provided by Gist Brocades (the Netherlands) and was used at 80 nkat ml-’ in 20 mM citrate phosphate buffer pH 4.5; this showed no PG activity under the conditions used. Pectic enzyme assays Enzyme activities were determined according to Versteeg et al. [33] for PME and Baron et al. [4] for PG, and have been described previously by Le Cam et al. [14]. Tissue degradation with enzymes Samples of 10 roots per cultivar were washed, sterilized in sodium hypochlorite solution (1 %) for 3 min, rinsed three times in sterile distilled water and air-dried. Three holes (7 mm depth) per root were excised with a punch (5 mm) and 20 pl of enzyme extract from M. acerina (28) containing 5 nkat of PG was placed in each hole. Twenty microlitres of sterile water was used as a control. After 48 h in a wet atmosphere at 30 “C, the degraded tissues were removed. The degree of tissue degradation was defined as the average weight of water which filled the holes. Preparation of cell wall material Cell walls were prepared as an alcohol-insoluble solid (AIS) according to Voragen et al. [34]. From 5 kg of roots, 200 g of tissue were taken randomly and cut into pieces, homogenized in 500 ml boiling aqueous 96 y. ethanol in a Waring blender for 25 s and stirred for 10 min. After filtration through a G3 sintered-glass filter, the AIS was washed with ethanol until the filtrate became colourless and gave a negative reaction in the phenol-sulphuric acid test [9] (to test for soluble carbohydrates). The residue was washed with acetone and ether, and air-dried. The average particle size was less than 1 mm. Chemical extraction of cell wall material For each AIS, the sequential extraction was carried out four times. AIS (1 g) was suspended in a solution (120 ml) of cyclohexane-diamino-tetracetic-acid* (CDTA), 20 mM pH 5.0 at 25 ‘C and stirred mechanically for 30 min. After filtration through G3 sintered-glass filter, the residue was resuspended in fresh CDTA solution (three extractions). The final residue was washed with distilled water. The pooled extracts containing soluble pectic fractions (CSP) were concentrated under vacuum, extensively dialysed (12000 mol.wt cutoff) against deionized water at 4 “C, then freeze-dried. This lyophilized fraction was dissolved in 50 mM sodium acetate buffer, pH 5-O and purified by anion-exchange chromatography. The CDTA insoluble residue was resuspended in 50 mM HCL (120 ml) and heated for 30 min at 85 “C. The suspension was filtered through a G3 sintered-glass filter. The extraction was then repeated twice and the final residue was washed with distilled water. All the extracts containing the

142

B. le Cam, P. Massiot,

C. Campion

and F. Rouxel

HCL soluble pectic fraction (HSP) were pooled and brought to pH 4.5 with a solution of 1 M sodium hydrogen carbonate, and treated as the CSP fraction. Enzymatic hydrolysis of cell wall polysaccharides Carrot AIS (10 mg) were suspended in 5 ml of 50 mM sodium acetate buffer pH 45 in glass tubes litted with Teflon-lined screw caps. M. acerina enzymes containing 0.5 nkat of PG activity were added and the suspension was stirred at 40 “C. After reaction for 15 and 30 min, the suspension was heated at 95PC for 5 min, centrifuged at 450g for 5 min and then filtered on 3 pm Millipore. filters. Filtrates were analysed for galacturonic acid using hydroxydiphenyl method [28]. Controls were performed without enzymes. CSP pectic fractions (1 ml containing 5 pmol of galacturonic acid in 50 mM sodium acetate buffer pH 4.5) were stirred in glass tubes, fitted with Teflon-lined screw caps, with endoPG (1 nkat) alone, or with a mixture of endoPG (1 nkat) and PME (1 nkat), or with M. acerina enzymes containing 1 nkat of PG activity. After reaction at 40 “C, the tubes were treated as above and the filtrates were analysed by high performance gel-permeation chromatography (HPGPC). Analytical methods AIS were ground (3 min) in a Retsch MM2 mixer mill. The neutral sugar composition was determined by gas chromatography (capillary column of 30 m x 0.25 mm i.d. with DB225, 0.15 pm film thickness; J&W Scientific), at 215 “C using hydrogen as the carrier gas, after sulphuric acid hydrolysis [2.5] and derivatization to alditol acetates [12]. Myo-inositol was used as the internal standard. Galacturonide content was estimated calorimetrically at 520 nm with m-hydroxydiphenyl [5] following sulphuric acid hydrolysis. [I]. In the soluble fractions, galacturonic acid and neutral sugars (expressed as arabinose) were determined by the automated m-hydroxydiphenyl [29] and orcinol [31] methods, respectively, the latter being corrected for interfering galacturonic acid. The degree of methoxylation, calculated as the molar ratio of methanol to galacturonic acid, was estimated by determining the methanol content released after alkaline hydrolysis of pectins. Methanol was measured by gas chromatography (Carbowax capillary column of 50 m x 0.32 mm i.d., 0.2 pm film thickness), at 70 “C using hydrogen as the carrier gas, after extraction by steam distillation from pectic solution (2 mg ml-‘) in 0.2 M NaOH for 1 h at room temperature. Propanol was used as the internal standard. Nitrogen was determined according to Moll et al. [20] and protein content was estimated as N x 6.25. High performance ion-exchangechromatography (HPIEC) A base anion-exchange column TSK DEAE 5PW (75 x 7.5 mm, Toyosoda, Japan) was connected to an HPLC system (Kontron). The column was eluted with 0.05 M sodium acetate pH 6.0 for 10 min, followed by a linear gradient 0.05-0.5 M of sodium acetate pH 6.0 for 47 min at a flow rate of 0.6 ml min- ‘. Samples of 200 1.11containing 5 l.trnol of galacturonic acid were injected. The eluate was continuously monitored at 520 nm using the automated m-hydroxydiphenyl method [29].

Carrot

cell wall polysaccharides

and susceptibility

to M. acerina

143

HPGPC

The molecular weight distribution of polysaccharides was determined using an HPLC system involving a Laboratory data control programmable pump, equipped with four Bio-Gel TSK columns (each 300 x 7.8 mm) in series (50,40,30 and 25 PWXL; BioRad Labs), in combination with a TSK XL guard column (40 x 6 mm) at 40 “C. The eluent was 0.4 M acetic acid/sodium acetate pH 3.6, used at a flow rate of O-8 ml min-‘. The eluate was continuously monitored at 520 nm using the automated mhydroxydiphenyl method [29]. Preparative

chromatography

Solutions (400 ml) of CSP or HSP (1 mg ml-‘) were each loaded onto a column (2.5 x 12 cm) of DEAE-Trisacryl (IBF) and eluted with 50 mM sodium acetate buffer pH 5-O at a flow rate of 35 ml h-‘. Fractions (10 ml) were assayed for galacturonic acid and neutral sugars. Pectic material bound on the gel was eluted with sodium acetate buffer pH 5.0 of appropriate ionic strength, calculated from HPIEC analysis. Purified pectin was then extensively dialysed against water and freeze-dried. Viscosily measurements

Intrinsic viscosity was obtained at 30 “C by measuring the flow times of solutions of pectins in 155 mM NaCl and 5 mM EDTA in an automatic capillary (0.46 mm) Ubbelhode viscosimeter (Viscologic TI 1, SEMATech) and by the double extrapolation to zero concentration based on the Huggins & Kramer equations [2]. Statistical

analysis

Analysis of variance and comparison of means were applied to the experimental with Newmans and Keuls’ test in procedures of SAS-STAT software [2S].

data

RESULTS

of carrot cultivars The carrot cultivars showed different susceptible to M. acerina and Major The response of Boltex towards M.

Relative susceptibility

to M. acerina

disease indices (Table 1). Touchon was the most and Flakkee were the least susceptible cultivars. acerina was intermediate.

Carrot tissue resistance to enzymes produced by M. acerina The degrees of tissue degradation (Table 1) enabled the cultivars to be ranked in the same sequence as the disease index. However, only Touchon was significantly more susceptible to enzymes from M. acerina than the other cultivars. Cell wall composition

The AIS from the four carrot cultivars made up between 3.0 and 43 y0 of the fresh wt and between 30.1 and 32 o/o of the d. wt of the tissue (Table 2). The cell wall content was not correlated with the relative resistances of the cultivars. The galacturonic acid content (Table 2) permitted division of the cultivars into two groups: Major, with 26 y. of AIS and Flakkee, Boltex and Touchon, with about 30 y. of AIS. Except for palactose and glucose,. there were no differences between the

B. le Cam, P. Massiot,

144

TABLE The

reaction

and F. Rouxel

1

of carrot cultivars of

C. Campion

fowards Mycocentrospora tissue degradation with erqmes

acerina produced

(isolate 28) : disease index, . by de fkrgus

degree

Cultivan

’

Disease index (mm*) Degree of tissue degradation (mg)

Flakkee

Boltex

143 (Zl)*a$

143 (13)a

205 (l8)b

226 (17)~

193 (39)ta

204 (36)a

234 (34)a

350 (59)b

*Mean of 40 samples (SD). tMean of 30 samples (SD). ,CVahtes followed by the same Keuls’ test, a = 805.

letter

do not differ

TABLE field

and composition

of

Touchon

Major

alcohol-insolrrble susceptibilities

significantly

to Newman

and

2

f rom f lo Mycocentrospora solid

according

(MS)

oar

carrot

callivars

exhibiting

d#eren f

acerina

Cultivars Major Yield Galacturonic Rhamnose Arabinose Xylose Mannose Galactose Glucose Total neutral Proteins

acid

sugars

*Expressed as a percentage IExpressed as a percentage ‘,mg g-’ of AIS.

3.4* 32.01 261: 23 76 12 17 142 149 419 89

Flakkee 43 387 300 24 49 15 17 71 145 321 87

Boltex 3.3 301 298 23 70 I8 20 94 154 379 97

Touchon 3.0 31.0 301 25 66 I4 17 II3 151 386 81

of the fresh wt of the tissue. of the d. wt of the tissue.

cultivars in the contents of the neutral sugars. In the group including Touchon, Boltex and Flakkee, galactose content was low compared to that of Major. Cellulose ant protein contents were similar in all cultivars. Characterization

of peetic fractions

The recovery of the purified pectins from the four carrot cultivars (Table 3) indicated that CSP contents were similar in Major, Flakkee and Boltex and lower than in Touchon, whereas HSP contents increased with the resistance of the cultivars. CDTA is a powerful chelating agent and appears to solubilize mainly pectic substances from the middle lamella, while hot hydrochloric acid solubilizes protopectin from the primary cell wall [27]. The CSP fractions from Major, Flakkee and Boltex contained

Carrot

cell wall Composition

polysaccharides

and susceptibility

145

to M. acerina

TABLE 3 of pectic fractions extracted from alcohol-insoluble solids exhibiting d$rent susceptibilities to Mycocentrospora

(AIS)

from four acerina

carrot cultivars

Cultivars Flakkee

Major Pectic

fractions

Yield Galacturonic acid Rhamnose Arabinose Xylose Mannose Galactose Glucose Molar ratio (gal A: rha) Degree or methoxylation

(%)

CSP

HSP

108 (5)* 80.57 2.4 64 0 0 10.5 0.2 33.5 63.9

184 (11) 47.1 7.2 42 0.3 0 35.8 5.4 65 15.9

CSP, Cyclohexan-diamino-tetracetic rha, galacturonic acid: rhamnose. *mg g-’ of AIS; mean of four tmol per 100 mol.

CSP

HSP

CSP

99 (6) 176 (10) 110 (4) 848 1.2 65 0 0 7.5 0.0 70.7 5D9

acid soluble replicates

Boltex

65.5 8.8 3.6 83 84 21.4 0.5 7.4 31.7

pectin;

849 1.3 5.0 0 0 8.6 82 653 680 HSP,

HCI

Touchon HSP

CSP

HSP

166 (15) 63.9 8.6 47 0 3.6 192 0 7.4 35.4

145 (4) 68.9 2.3 142 0 0 146 @O 380 66.7

103 (13) 57.6 65 5.2 0 1.8 28.1 0.8 8.9 23.1

soluble

pectin;

Gal A:

(SD).

a large amount of galacturonic acid (80-85 %) compared to Touchon (68.9 %). The molar ratio galacturonic acid : rhamnose in CSP from Flakkee and Boltex were higher (two-fold) than those of Major and Touchon. Galactose and arabinose contents were higher in Touchon than in other cultivars. The composition of HSP fractions showed that Major contained 47.1 o/0 galacturonic acid, while the others contained between 57.7-65.5 %. Galactose content, the second most common compound in the HSP fraction, varied from 19.2-35.8 %. Compared to the CSP fractions, the HSP fractions showed low galacturonic acid: rhamnose molar ratios suggesting the presence of arabinogalactan linked to the rhamnogalacturonan backbone, probably through rhamnosyl residues, which are the branching points of the side chains [19]. The degrees of methoxylation (DM) of Major and Touchon were not significantly different (64-67 %) between the CSP fractions. The DM of the HSP fraction was lower than that for the CSP fraction, but varied between the cultivars, the lowest value obtained for Major. Theintrinsic viscosities and the viscosity-average molecular weights (Mv) of the CSP fractions are given in Table 4. Mv values of the pectins purified from Major and Touchon were similar and lower than in the other two; the value for Boltex was higher. Gel-filtration chromatograms of CSP fractions (Fig. 1) showed an homogenous distribution of galacturonic acid-containing polysaccharides. The elution times of the peaks corresponded to the Mv values, except for the Boltex pectin. Enzymatic hydrolysis of cell wall material The AIS from the four carrot cultivars were hydrolysed with the enzymes produced by two isolates of M. ace&a (Table 5). In previous work [14], the aggressive isolate

B. le Cam, P. Massiot,

146

TABLE

C. Campion

and F. Rouxel

4

Intrinsic viscosities and vticosiry-average molecular weights (Mu) of cyclohexan-diamino-t&acetic soluble pectinsfrom four carrot cultivars exhibiting dl$rent susceptibilities lo Mycocenerospora

acid acerina

Cultivars

Intrinsic Mv

viscosity

(ml g-‘)

Major

Flakkee

Boltex

191 38000

235 44006

310 54500

015

Elution

Fro 1. High performance gel-permeation cyclohexan-diamino-tetracetic acid soluble susceptibilities to A4ycocenfrospora acerina.

Solubilization

Touchon 195 38000

time (min)

chromatography pectin from four

analysis of the size distribution of carrot cultivan exhibiting different

TABLE 5 rate of pectins from alcohol-insoluble solid (AIS) f rom f our carrel cultivars fhe eqymes produced by two isolates of Mycocentrospora acerina

aJer exposure of

Cultivars Major Aggressive isolate (28) Hypoaggressive isolate *Expressed the maximum

(30)

as pmol of galacturonic error was +5 %.

Flakkee

26* 1.9

Boltex

3.4 2.0 acid min-’

and g-’

41 3.3 of AIS;

mean

Touchon 4.7 3.5 of two analyses

and

exhibited higher solubilization rates of pectins than the hypoaggressive isolate. The ranking of the cultivars was similar with the two isolates. Pectins from the most susceptible cultivars, Boltex and Touchon, were hydrolysed and solubilized more extensively than those from the other cultivars. The solubilization rate was the lowest with Major, the cultivar least susceptible to M. acerina.

Carrot

cell wall polysaccharides 20

and susceptibility

147

to M. acerina

15-

Hypoaggressive FIG 2. Variation of the elution volume (Ve) in high performance graphy of cyclohexan-diamino-tetracetic acid soluble pectins (CSPs) Touchon (0) after 15 min of hydrolysis with enzymes produced [aggressive (28) and hypoaggressive (30) isolates]. Bars represent two variation was expressed as Ve o/0 Ve of initial CSP fraction.

25-

-

PME

s -

20s % 158 ‘3 .; s

+ endoPG

isolal gel-permeation chromatofrom cvs Major ( n ) and by Mycocenfrospora acerina standard deviations. The

-

--t

lo-

+

5-

endoPG

0

45

15 Time

of hydrolysis

30

45

(min)

FIG 3. Variation of the elution volume (Ve) in high performance gel-permeation chromatography of cyclohexan-diamino-tetracetic acid soluble pectins (CSPs) from cvs Major (B) and Touchon (0) after hydrolysis with endopolygalacturonase (endoPG) and pectin methylesterase (PME). Bars represent two standard deviations. The variation was expressed as Ve%Ve ofinitial CSP fraction.

Enzymatic

hydrolysis

of pectic fractions

The CSP fractions from the cultivars most (Touchon) and least (Major) susceptible to M. acerina infection were hydrolysed with enzymes produced by the two isolates of M. acerina and analysed in the HPGPC system (Fig. 2). After 15 min incubation, there was an increase in the elution volumes (Ve) of the CSP fractions, indicating a decrease of the hydrodynamic volume of the molecules. With both isolates, the variation of Ve of the Touchon CSP was twice that of Major CSP, indicating that hydrolysis of Touchon pectin was the most extensive. Otherwise, the variations of Ve-were more important with the aggressive than with the hypoaggressive isolate, which agrees with results on enzymatic hydrolysis of AIS (Table 5). The CSP fractions were incubated with pure endoPG and PME (Fig. 3). After 45 min of reaction with endoPG alone, Touchon pectin was hydrolysed whereas Major pectin was not. Addition of PME induced a depolymerization of the Major pectin and an increase of Ve of the Touchon pectin. After 45 min reaction with endoPG and

B. le Cam, P. Massiot,

148

C. Campion

and F. Rouxel

Touchon I----

0

I

I

I

20

40

60

80

0

I

I

I

20

40

60

80

Elution time (min) Elution time (min) FIG 4. High performance ion-exchange chromatography of cyclohexane-diamino-tetracetic acid soluble pectins from cvs Major and Touchon after 45 min hydrolysis with endopolygalacturonase (endoPG) and pectin methylesterase (PME).

PME, the variation in Ve was five times higher than with the endoPG alone. These results indicated that PME action allowed the release of sites for endoPG action, especially in the Major pectin. The elution patterns of CSP fractions in HPIEC (Fig. 4) showed, only in the case of Touchon pectin, that endoPG activity produced a peak corresponding to a charged polymer. Addition of PME to endoPG produced a shift of the main peak towards increased ionic strengths for both cultivars and a disappearance of the charged peak on the Touchon chromatogram. DISCUSSION

The results of this study indicated that the ranking of susceptibility of the carrot cultivars tested to the phytopathogenic fungus M. acerina was the same as the rank for tissue maceration with the pectinolytic enzymes from M. ace&z. There was a significant difference

between

the degree

of tissue degradation

of Touchon,

the most susceptible

cultivar, and the others. Moreover, the’hydrolytic ability of pectic substances in the cell wall material appeared to be linked to the relative resistances of the cultivars, especially for the two extreme cultivars; at the beginning of hydrolysis, the solubilization rate of the pectins from the most susceptible cultivar was l-8 times higher than that of the least susceptible, with both isolates. These results differ from those of Valsangiacomo et al. [3.7], who found no correlation between the expression of resistance to Venturia inuequulis and in vitro degradability of the cell walls of apple leaves. However, the tested commercial enzyme preparations they used, appeared to be very active on cell wall polysaccharides but PG isolated from V. inut?qualiswas not efficient enough to release pectins. The yield of cell wall materials expressed as AIS, and their composition, agree with the previous studies on carrot cell walls [17,.?8,34]. Similar results were obtained by Robertson et al. [23,24] who showed that the amounts of cell wall material represented from 3.2 to 4.6 Y. of the fresh wt, depending on the cultivars. In contra& to the results

Carrot

cell wall polysaccharides

and susceptibility

to M. acerina

149

ofPage & Heitefuss [22] obtained with potato tubers, we found no positive correlation between the amount of cell wall material, the polygalacturonan content and the degree of resistance. On the contrary, the galacturonic acid content of AIS and the amounts of extracted pectins were higher in the most susceptible cultivars, Boltex and Touchon. Hondelmann & Richter [13] showed that the susceptibility of strawberries to Botrytis cinerea was highly correlated with the soluble pectin content of the fruit; other authors suggested that the insoluble form of pectins was more resistant to hydrolysis by pectinolytic enzymes of plant pathogens [la, 3.51. In accordance with these results, the amounts of HSP fractions from carrot, corresponding to the insoluble pectin, protopectin, increased with the resistance of the cultivars. The cultivars showed no major differences in the composition of their pectic the presence of highly esterified, slightly branched fractions. This confirmed rhamnogalacturonan in the CSP fraction, corresponding to the pectins of the middle lamella [27], and the presence of highly branched rhamnogalacturonan in the HSP fraction, corresponding to the pectins of the primary cell wall [27]. The degrees of methoxylation of the carrot pectins agrees with other reports in the literature [17,28, 343, but they do not allow discrimination between the cultivars, in contrast to previous data [l&22,34] which reported a slight correlation between the degree of pectin esterification of potato and the resistance of potato tubers to Erwinia carotouora. It was suggested that the cross-linking of poorly esterilied galacturonan chains with calcium conferred resistance to enzymatic hydrolysis [22,30-j. Our data indicate that the esterified polygalacturonan content of the susceptible cultivars was slightly but not significantly higher than that of the other cultivars. The differences in susceptibility of carrot cultivars and in pectin content do not allow any firm conclusions to be made on the relationship between the composition of pectic polysaccharides and resistance to M. acerina. Nevertheless, the CSP:HSP ratio seemed positively linked to the relative susceptibilities of cultivars to infection. The enzymatic hydrolysis of pectic fractions (CSP) with enzymes from M. acerina confirmed that the aggressiveness of isolates was correlated with the activity of pectinolytic enzymes in the pathogen [14]. For a given isolate, we observed a difference in the enzymatic breakdown of the pectic polysaccharides between the two extreme cultivars, corresponding to a difference in structure, in spite of their similar chemical compositions. The difference in behaviour of endoPG on pectin (CSP) from the Touchon and Major cultivars indicated a different distribution of methyl groups along the rhamnogalacturonan chains, although the degrees of methoxylation were similar. The pectin of the most susceptible cultivar, Touchon, presented sites of action for depolymerization, whereas those of Major did not. The addition of PME involved the removal of the methoxyl groups thus enhancing the action of endoPG as previously observed in orange pectin [3]; in this case, the structural difference was suppressed. Although the degrees of methoxylation were similar, we suggest that the repartition of the esterified galacturonic acid units was different. As we did not detect any pectin lyase activity [14], and a late secretion of pectate lyase in infected tissue (unpublished data), this structural difference could explain the variation in tissue maceration. Furthermore, PME was detected [14], but with an optimum activity at pH 7, much higher than the carrot tissue pH (5.5) [16], suggesting the importance of PG action in the early critical stages of M. acerina pathogenesis.

6. le Cam, P. Massiot,

150

C. Campion

and F. Rouxel

Other structural features such as the nature and the distribution of the side chains, the cross-linking of pectic polysaccha.rides with other cell wall polymers, might modify pectin hydrolysis by fungal enzymes. Structural analyses will be continued in order to explain the relative resistance of some pectins from the middle lamella to enzymatic breakdown, a feature which appears to be widely linked to cultivar susceptibility to M. ace&a.

We wish Sinoquet technical Barfleur,

to thank Dr J. F. Drilleau for his critical comments on the manuscript, Dr E. for the gift of endopolygalacturonase an14 N. Marnet and C. Prioult for their assistance. This research was supported by a grant from the SILEBAN, 50760 France.

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