Pectolytic enzymes produced in vitro and duringcolonization of melon tissues by Didymella bryoniae

G. Chilosi and P. Magro*†Dipartimento di Protezione delle Piante, Universita degli Studi della Tuscia, Via S. Camillo de Lellis, 01100 Viterbo, Italy

Pectolytic enzymes produced by Didymella bryoniae in a liquid medium containing pectin as sole carbon source and ininoculated etiolated hypocotyls of 10 melon cultivars, as well as those constitutively expressed in spores, were studiedby isoelectric focusing, quantitatively and qualitatively. Five constitutive pectin lyase (PNL) isoenzymes differing inisoelectric joint (pI), one acidic (pI 3·9) and four basic (pI 8·4, 8·9, 9·3, 9·9) were expressed in extracts from spores.The same PNL isoenzyme pattern was detected in culture filtrates and in infected tissues of all the melon cultivarstested. Polygalacturonase (PG) activity, represented by a single inducible acidic band (pI 4·6) was detected only inculture filtrates. A single constitutive basic pectin methylesterase (PME) isoenzyme (pI> 10·0) was also found inspores, culture filtrates and inoculated melon tissues. All cultivars were susceptible at the seedling growth stage, butwith differences in disease severity; cultivars Amarillo Oro and Juane Canari were, respectively, the least and mostsusceptible. Pectin lyase activity was highly correlated with disease severity. In rotted tissues and culture filtrates, anincrease in pH to values over 7·0 was recorded, values optimal for PNL activity. In this plant–pathogen interaction,PNL activity represents the principal pectolytic component and these isoenzymes were associated with the onset ofdisease, disease severity and an increase in pH of infected tissue.

Introduction

Many plant pathogenic fungi are known to produce arange of cell wall degrading enzymes that macerate plantcell walls, including pectolytic enzymes such as pectinmethylesterase (PME), polygalacturonase (PG) and pectinand pectate lyase (PNL and PL, respectively) that mayhave important roles in the infection process and in thedevelopment of disease symptoms (Bateman & Basham,1976; Walton, 1994). Pectolytic enzymes are oftenproduced in culture and during plant infection sequen-tially as multiple isoenzymes and may constitute acatabolic pathway for the complete degradation ofpectic polysaccharides, the process being initiated byconstitutive forms (Leone & Van Den Heuvel, 1987;Chilosi & Magro, 1997). The importance of suchenzymes in pathogenicity is supported by the ability ofpurified enzymes to reproduce disease symptoms (Bate-man, 1968; Marciano et al., 1982; Barash et al., 1984;Holtz & Knox-Davies, 1985) and by the correlation ofthe pectolytic enzyme level with the extent of damage tothe plant (Olsson, 1989; Wijesundera et al., 1989; Cleve-land & Cotty, 1991; Baayen et al., 1997). Moreover,evidence of PL and PNL involvement in pathogenesis has

been obtained using hypovirulent isolates (Marcus et al.,1986), nonpathogenic mutants (Wattad et al., 1995),antibodies blocking enzymes (Crawford & Kolattukudy,1987; Wattad et al., 1994). However, disruption of a PNLgene of Glomerella cingulata did not result in reducedvirulence (Bowen et al., 1995).

Didymella bryoniae (anamorph Ascochyta cucu-meris), the pathogen causing gummy stem blight (foliarphase) and black rot (fruit phase) of cucurbits, is animportant cause of losses in this family. Water-soakedirregular patches develop on leaves, and the lesions turnbrown. On fruits, lesions may appear sunken and rotted,but internal rot often develops without any externalsymptoms (Ebben, 1988). The type of symptom suggeststhat pectolytic enzymes might play a role in tissuecolonization by this pathogen, but to our knowledge noinformation is available on the involvement of theseenzymes in the development of the disease.

This paper describes a study of the capacity ofD. bryoniae to produce pectolytic enzymes in cultureand during infection of melon seedlings and of theoccurrence of constitutive pectolytic isoenzymes infungal spores.

Materials and Methods

Fungus and fungal culture

D. bryoniae isolate CP 1799, supplied by Professor

Plant Pathology (1998) 47, 700–705

700 Q 1998 BSPP

*To whom correspondence should be addressed.

†E-mail: magro@unitus.it

Accepted 29 June 1998.

E. de Neergaard, The Royal Veterinary and Agricul-tural University of Copenhagen, Denmark, was cul-tured on potato dextrose agar (PDA) (Oxoid Unipath,Basingstoke, England) at 258C. For the enzyme pre-parations, the isolate was surface cultured in Czapek’sliquid medium (pH 5·0) containing NaNO3 (2 g L¹1),KH2PO4 (1 g L¹1), MgSO4·7H2O (0·5 g L¹1), KCl(0·5 g L¹1), FeSO4·7H2O (0·01 g L¹1), ZnSO4·7H2O(0·01 g L¹1) and 10 g L¹1 of citrus pectin (Sigma,Chemical Co., St. Louis, USA) as sole carbon source.The inoculum was one agar disc (6 mm diameter) cutfrom the edge of 3-day-old cultures on PDA. Themycelium was grown at 258C in 250-mL Erlenmeyerflasks containing 50-mL medium.

Plant material

Melon seeds (Cucumis melo, cv. Amarillo Oro, Hale’sBest, Giallo Rugoso, Gold Star, Jaune Canari, Myras,Pepito, Retato degli Ortolani, Tendral Verde and Verded’Inverno) were surface-treated by immersion in H2O2

(90 g L¹1) for 15 min and rinsed in sterile water for 1 h.To obtain etiolated hypocotyls, seeds were then sown insterilized vermiculite, watered daily with sterilized waterand grown in the dark at 258C at approximately 95%RH for 6 days.

Inoculation and disease assessment

A spore suspension was prepared by washing 14-day-oldcultures on PDA with sterile water and adjusting it to25 ×104 spores mL¹1. Inoculum for seedlings wasobtained by evenly distributing 1 mL of spore suspensionon the surface of PDA (2 mm thick) in Petri dishes(90 mm diameter).

At 6 days old, etiolated hypocotyls were inoculated byplacing PDA blocks (4 ×2 mm) supporting the sporesuspension on the stem 5 cm below the cotyledons. Theseedlings were then placed in the dark at 258C and 95%RH for 48 h. For controls, seedlings of each cultivar wereinoculated with sterile PDA blocks. To check forcontaminants, some inoculated hypocotyls were asepti-cally placed into Petri dishes containing PDA, but onlyD. bryoniae grew under these conditions.

Disease was assessed 48 h after inoculation as meanlength (cm) of the lesions recorded for each seedling ofeach cultivar.

Enzyme extraction from fungal cultures andinoculated melon seedlings

At 2, 4, 6, 8 and 12 days after inoculation liquid fromthree flasks was collected and the mycelium wasremoved by filtration in a Buchner funnel. The culturefiltrates were tested for pH, centrifuged at 15 000 g for15 min at 48C and then the supernatants were dialysedagainst distilled water at 48C.

Enzyme preparations from inoculated seedlings wereobtained by grinding infected tissues in an ice-cooled

mortar in 0·05 M Tris-HCl buffer pH 7·8 (1g tissue mL¹1

buffer) containing 0·1 M KCl, 5 g L¹1 cysteine and10 g L¹1 insoluble polyvinylpolypyrolidone (Sigma). Theslurry was strained through three layers of cheesecloth,centrifuged at 15 000 g for 20 min at 48C and dialysedagainst several changes of distilled water at 48C. The sameprocedures were applied to uninoculated plant tissues.

Enzyme extraction from spores

Two-week-old D. bryoniae cultures were flooded withsterile water, and a glass rod was used to remove sporesgently from the surface of the cultures. The spores werethen collected and twice washed with sterilized distilledwater by centrifugation at 3500 g for 20 min at 48C. Thepellet was suspended in 5 mL of 0·1 M Na-acetate bufferpH 5·0, giving a concentration of approximately50 ×106 spores mL¹1, ground in an ice-cooled mortarfor 15 min and centrifuged at 15 000 g for 20 min at 48C.The supernatant was collected, dialysed against distilledwater at 48C and filter-sterilized through a 0·22-mmmembrane (Millipore, Bedford, USA). The supernatantwas then quickly frozen and lyophilized to dryness. Theresidue was redissolved in 0·5 mL of distilled water.

Pectolytic enzyme assays

PNL activity was assayed spectrophotometrically bymeasuring the increase of absorbance at 235 nm. Anincrease of 1·73 indicated the formation of 1 mmol ofunsaturated uronide (Zucker & Hankin, 1970). One unitof enzyme activity (PNLU) catalysed the formation of1mmol of unsaturated uronide min¹1 from 2·5 g L¹1 (w/v)citrus pectin in Tris-HCl buffer (0·1 M, pH 8·5) at 358C.

PG activity was determined by measuring the increaseof reducing-end groups over time by the method of Nelson(1944), using galacturonic acid (Sigma) as a standard. Oneunit of PG activity (RU) produced 1 mmol of reducinggroups min¹1 from 2·5 g L¹1 polygalacturonic acid(Sigma) in Na-acetate buffer (0·1 M, pH 5·0), at 358C.

Isoenzyme identification

Isoenzyme separation by isoelectric focusing (IEF) wasperformed horizontally on Multiphor II apparatus(Pharmacia Biotech, Uppsala, Sweden) by using 0·4 mmthick polyacrylayamide gels containing 5% (v/v) ampho-lytes (Pharmacia Biotech, Uppsala, Sweden) covering thepH range 3·5–10·0. The run was carried out at a constantpower of 5 W for 1·5 h. After IEF, polyacrylamide gelswere overlaid with ultrathin overlay gels (0·4 mm) forPNL and PG isoenzyme detection, prepared as describedby Ried & Collmer (1985) with the following modifi-cations: for PNL detection, the 10 g L¹1 agarose (Sigma)gel contained 1 g L¹1 pectin in 50 mM Tris-HCl buffer,pH 8·5; for PG detection the 10 g L¹1 agarose gelcontained 1 g L¹1 polygalacturonic acid and 10 mM

EDTA buffered at pH 5·0 with 50 mM Na-acetate. IEFpolyacrylamide gels overlaid with ultrathin agarose gels

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were incubated at 100% humidity and 358C for 30–60 min. Activity bands were visualized by staining theagarose overlay for 10 min in 0·5 g L¹1 ruthenium red(Sigma) solution, followed by rinsing in distilled water.PNLs or PGs appeared as white bands. According toCruickshank & Wade (1980) PME appeared as dark redbands. The pI values of pectolytic isoenzymes wereestimated from a regression equation of standardproteins (Pharmacia) vs. the distance migrated.

Statistical analysis

Data on disease severity were analysed using analysis ofvariance and least significance differences (LSD) werecalculated at P ¼ 0·05 using Bonferroni test levels. ThePearson coefficient of simple correlation (r) was calcu-lated between disease severity and PNL production onvarious infected cultivars.

Results

Production of pectolytic enzymes from fungalcultures and extract from spores

The time course of PG and PNL production byD. bryoniae grown on a medium containing pectin assole carbon source is shown in Fig. 1. The maximumPNL and PG activities were reached between 4 and8 days; from day 10, PNL and PG activity levelled off.During growth, the pH of the culture medium increasedto a maximum (pH 8·7) that coincided with themaximum of the PNL and PG activity. The 6-dayincubation time was therefore chosen to obtain culturefiltrates for further enzyme characterization.

The relative activity of PNL on polygalacturonic acid,as compared with pectin, was 68%; the relative activity

of PG on pectin, as compared with polygalacturonicacid, was 20%. The optimum temperature and pH forPNL was 358C and 8·5, respectively; that for PG was408C and 5·0 (Fig. 2).

PNL activity was enhanced when CaCl2 was added toreaction mixtures to give a concentration between 1 and20 mM, although there was significant activity in theabsence of added Caþþ.

In extracts from ground spores PNL activity wasdetected (0·2 PNLU mL¹1 spore extract), whereas nodetectable PG activity was observed.

Disease development, pectolytic activity and pH inmelon hypocotyl extracts

Inoculated and uninoculated hypocotyls were monitored48 h after inoculation for development of symptoms andassayed for PNL and PG activity. Water-soaked lesionswere observed in all the inoculated cultivars after 48 h,but with significant differences in disease severity values

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Figure 1 Time course of pectin lyase (W) and polygalacturonase (A)produced by Didymella bryoniae isolate CP 1799 in liquid mediumsupplemented with pectin as sole carbon source. Incubation time isexpressed in days. Pectin lyase and polygalacturonase activity areexpressed, respectively, as units and reducing units. For clarity onlythe upper error bars are given, which indicate the standard error ofthe means of three experiments.

Figure 2 (a) pH and (b) temperature effect on the activity of pectinlyase (PNL) (W) and polygalacturonase (PG) (A) produced by Didy-mella bryoniae isolate CP 1799 in liquid medium. Appropriate buffer-ing solutions (Na-acetate, Na-phosphate, Tris-HCl, 50 mM) were usedto cover the needed pH range (4·0–9·0): minimum interval, 0·5 units.Reaction mixtures were incubated in the 25–508C temperaturerange, with 58C intervals. Data are expressed as percentage of PNLand PG maximum activity.

(Table 1). PNL activity was detected in extracts from allthe cultivars tested (Table 1) but PG activity was notfound in any of the extracts. Increasing disease severityvalues were reflected in increasing PNL activities (Table1) with a linear correlation of r ¼ 0·907 (Fig. 3). Controlplants treated with sterile PDA blocks remained healthy,with no PNL nor PG activity in extracts from tissues.

The pH of exudates from inoculation sites wasbetween 6·2 and 6·4 for controls, and over 7·30 forrotted tissues.

PNL and PG isoenzyme patterns

The dialysed extracts from ground spores, culturefiltrates and extracts from infected melon seedlingswere subjected to thin-layer polyacrylamide gel IEF and

evaluated for PNL and PG isoenzymes. One PG bandwith pI 4·6 was observed in the culture filtrate (Fig. 4). Inaccordance with the results obtained from the quantita-tive PG assay, no PG isoenzymes were detected in extractsfrom ground spores or from infected melon cultivars. Fiveconstitutive PNL isoenzymes were found from ungermi-nated spores and culture filtrates, one being acidic (PNL1,pI 3·9) and four basic (PNL2, pI 8·4; PNL3, pI 8·9; PNL4,pI 9·3; PNL5 pI 9·9) (Fig. 5). The same constitutiveisoenzymatic pattern was detected in extracts of all themelon cultivars (Fig. 5). PNL5 appeared to be super-imposed by a dark red band that indicated the possiblepresence of a PME isoenzyme with a pI>10·0. This PMEisoenzyme was also detected from spores, in vitro and inall the cultivars tested, thereby indicating its constitutiveexpression (Fig. 5). Acidic PNL1 appeared to be the majorPNL band compared with basic bands characterized by asimilar intensity. No PNL bands were detected in extractsfrom control seedlings.

Discussion

D. bryoniae produced pectin lyase both in vitro and ininfected melon hypocotyls. The optimum pH andtemperature of PNL and PG activity and Caþþ require-ment of PNL activity were comparable to those reportedfor corresponding enzymes from other fungi (Magro,1984; Wijesundera et al., 1984; Movahedi & Heale,1990; Bugbee, 1990; Johnston & Williamson, 1992;

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Table 1 Pectin lyase activity in extracts from hypocotyls of 10melon cultivars inoculated with Didymella bryoniae isolate CP 1799and disease severity on the cultivars: within columns, values fol-lowed by the same letter are not significantly different (P < 0·05)

RCultivar Disease severity* Pectin lyase activity**

Amarillo Oro 1·13 a 0·30 aHale’s Best 1·14 a 0·35 bcdMyras 1·20 ab 0·32 abTendral Verde 1·25 ab 0·36 bcdRetato degli Ortolani 1·27 ab 0·34 abGold Star 1·38 abc 0·35 bcdPepito 1·51 abc 0·37 bcdGiallo Rugoso 1·69 bc 0·39 dVerde D’Inverno 1·79 c 0·43 eJaune Canari 1·85 c 0·46 e

*Disease severity is expressed as mean of length (cm) of the lesionsrecorded for each melon seedling of the same cultivar infected withDidymella bryoniae.**Activity in units per gram fresh weight of extracted melon seedlingsinfected with Didymella bryoniae.

Figure 3 Correlation between pectin lyase activity (PNL units pergram of infected tissue) in seedlings of 10 melon cultivars inoculatedwith Didymella bryoniae isolate CP 1799 and disease severity.

Figure 4 Polygalacturonase isoenzyme pattern from culture filtrate ofDidymella bryoniae isolate CP 1799 grown on in liquid medium sup-plemented with pectin as carbon source. Sample was separated onan isoelectric focusing (IEF) gel (pH 3·5–10·0), followed by agaroseoverlay activity staining. Position of polygalacturonase band is indi-cated on the right. Position and pI values of IEF markers are shownon the left.

Chilosi & Magro, 1997). IEF showed that one acidic andfour basic PNL isoenzymes and one basic PME wereconstitutively expressed in ungerminated spores,whereas no PG isoenzymes were detected. The PNL andPME isoenzymatic patterns from spores were alsoproduced by the pathogen in culture filtrates and ininfected tissues of the various cultivars. PNL activitydetected in inoculated tissues appeared to be of fungalorigin, since no bands were found in healthy tissues. PGappeared to be produced in culture filtrates only as a singleisoenzyme. This shows that, under the experimentalconditions described, this enzyme is induced only invitro. All cultivars tested were colonized by the fungus butwith significant difference in disease severity. Moreover,during colonization, linear increases of disease wereaccompanied by increasing PNL production, indicating apossible causal relationship. These results indicate thatPNLs produced by D. bryoniae may play a central roleduring pathogenesis in melon. Hancock (1966) showedsimilarly that Colletotrichum trifolii produced pectatelyase as the principal pectolytic enzyme, and raised the pHon different media. PNL was also the predominantpectolytic enzyme produced by Rhizoctonia solani (AG2strain) in culture and in infected sugar beet crowns(Bugbee, 1990), and PNL and PG were produced in vitro

by Colletotrichum lindemuthianum, but only PNL wasdetected in infected bean hypocotyls (Wijesundera et al.,1989). We observed an increase in pH during the in vitrogrowth of D. bryoniae, and in infected tissues to a levelclose to the PNL optimum that might have enhanced thePNL activity and the pathogenicity of this fungus. The pHincrease in the culture medium during fungal growth mayhave been caused by differential uptake of cations andanions. Transport of anions such as phosphate may act asthe hydroxide exchange system with the medium becom-ing more basic (Griffin, 1994). The pH increase in tissuemay be related to production of ammonia during de-amination of L glutamate catalysed by glutamate dehy-drogenase and of phenylalanine by phenylalanine ammo-nia lyase (PAL) (Wijesundera et al., 1989). It is wellestablished that oligogalacturonide fragments from plantcell walls produced by pectolytic enzyme activity inducerapid responses that activate plant defensive reactions.These responses include extracellular alkalinization andan oxidative burst, as found in tobacco cultured cellstreated with oligogalacturonides and PNL from Aspergillusjaponicum (Mathieu et al., 1996; Rouet-Mayer et al., 1997).

It is possible that D. bryoniae does not produceorganic acids such as oxalic acid in vitro or duringhost colonization as do other plant pathogens (Bateman& Beer, 1965; Maxwell & Lumsden, 1970; Marcianoet al., 1983; Magro et al., 1984). Thus, pathogenesis maybe induced as a consequence of the increase in pH of thehost tissue that permits the PNLs produced by the fungusto be more active. On present results, and on those ofother authors, the importance of fungal pectolyticenzymes in pathogenesis has to be considered case bycase according to the specific host–pathogen interaction.However, the ability of the pathogen to modify the pHlevel in planta during infection seem to be one of thecritical components.

Acknowledgements

We thank Dr B. Williamson (Scottish Crop ResearchInstitute, Invergowrie, Dundee, Scotland) for criticalreading of the manuscript.

The research was supported by the Ministero per lePolitiche Agricole (MIPA), Piano Nazionale Biotecnologie,Project n. 152.

References

Baayen RP, Schoffelmeer EAM, Toet S, Elgersma DM, 1997.Fungal polygalacturonase activity reflects susceptibility ofcarnation cultivars to fusarium wilt. European Journal ofPlant Pathology 103, 15–23.

Barash I, Zilberman E, Marcus L, 1984. Purification of Geo-trichum candidum endopolygalacturonase from culture andfrom host tissue by affinity chromatography on cross-linkedpolypectate. Physiological Plant Pathology 25, 161–9.

Bateman DF, 1968. The enzymatic maceration of plant tissue.Netherlands Journal of Plant Pathology 74, 67–80.

Bateman DF, Basham HG, 1976. Degradation of plant cellwalls and membranes by microbial enzymes. In: Heitefuss

G. Chilosi & P. Magro704

Q 1998 BSPP Plant Pathology (1998) 47, 700–705

Figure 5 Didymella bryoniae isolate CP 1799 pectin lyase (PNL) andpectin methyl-esterase (PME) isoenzyme patterns from spores (A),from culture filtrates (B), from etiolated melon hypocotyls, cultivarAmarillo Oro (C). Samples were separated on an isoelectric focusing(IEF) gel (pH 3·5–10·0), followed by agarose overlay activity staining.Positions of pectin lyase and pectin methyl-esterase bands are indi-cated on the right. Position and pI values of IEF markers are shownon the left.

R, Williams PH. eds Physiological Plant Pathology. Berlin,Germany: Springer-Verlag, 316–355. (Pirson A, Zimmer-mann MH, eds Encyclopedia of Plant Physiology NewSeries; vol. 4.).

Bateman DF, Beer SV, 1965. Simultaneous production andsynergistic action of oxalic acid and polygalacturonaseduring pathogenesis by Sclerotium rolfsii. Phytopathology55, 204–11.

Bowen JK, Templeton MD, Sharrock KR, Crowhurst RN,Rikkerink EHA, 1995. Gene inactivation in the plantpathogen Glomerella cingulata: three strategies for the dis-ruption of the pectin lyase gene pnlA. Molecular and Gen-eral Genetics 246, 196–205.

Bugbee WM, 1990. Purification and characteristics of pectinlyase from Rhizoctonia solani. Physiological and MolecularPlant Pathology 36, 15–25.

Chilosi G, Magro P, 1997. Pectin lyase and polygalacturonaseisoenzyme production by Botrytis cinerea during the earlystages of infection on different host plants. Journal of PlantPathology 78, 61–9.

Cleveland TE, Cotty PJ, 1991. Invasiveness of Aspergillusflavus isolates in wounded cotton bolls is associated withproduction of a specific fungal polygalacturonase. Phyto-pathology 81, 155–8.

Crawford MS, Kolattukudy PE, 1987. Pectate lyase fromFusarium solani f.sp. pisi: purification, characterization, invitro translation of the mRNA, and involvement in patho-genicity. Archives of Biochemistry and Biophysics 258,196–205.

Cruickshank R, Wade GC, 1980. Detection of pectic enzymesin pectin-acrylamide gels. Analytical Biochemistry 107,177–81.

Ebben MH, 1988. Didymella bryoniae (Auersw.) Rehm. In:Smith IM, Dunez J, Lelliot RA, Phillips DH, Archer SA edsEuropean Handbook of Plant Diseases. Oxford, UK: Black-well Scientific Publications, 349–350.

Griffin DH, 1994. Fungal Physiology. New York, USA: Wiley-Liss, Inc.

Hancock JG, 1966. Pectate lyase production by Colletotri-chum trifolii in relation to changes in pH. Phytopathology56, 1112–3.

Holtz G, Knox-Davies PS, 1985. Production of pectic enzymesby Fusarium oxysporum f.sp. cepae and its involvement inonion bulb rot. Phytopathologische Zeitschrift 112, 69–80.

Johnston DJ, Williamson B, 1992. Purification and characteri-zation of four polygalacturonases from Botrytis cinerea.Mycological Research 96, 343–9.

Leone G, Van den Heuvel J, 1987. Regulation by carbohy-drates of the sequential in vitro production of pecticenzymes by Botrytis cinerea. Canadian Journal of Botany65, 2133–41.

Magro P, 1984. Production of polysaccharide-degradingenzymes by Septoria nodorum in culture and during patho-genesis. Plant Science Letters 37, 63–8.

Magro P, Marciano P, Di Lenna P, 1984. Oxalic acid produc-tion and its role in pathogenesis of Sclerotinia sclerotiorum.FEMS Microbiology Letters 24, 9–12.

Marciano P, Di Lenna P, Magro P, 1982. Polygalacturonaseisoenzymes produced by Sclerotinia sclerotiorum in vivoand in vitro. Physiological Plant Pathology 20, 201–12.

Marciano P, Di Lenna P, Magro P, 1983. Oxalic acid, cell wall

degrading enzymes and pH in pathogenesis and their signif-icance in the virulence of two Sclerotinia sclerotiorum iso-lates on sunflower. Physiological Plant Pathology 22, 339–45.

Marcus L, Barash I, Sneh B, Koltin Y, Finkler A, 1986. Purifi-cation and characterization of pectolytic enzymes producedby virulent and hypovirulent isolates of Rhizoctonia solaniKuhn. Physiological and Molecular Plant Pathology 29,325–36.

Mathieu Y, Sanchez FJ, Droillard MJ, Lapous D, Lauriere C,Guern J, 1996. Involvement of protein phosphorylation inthe early steps of transduction of the oligogalacturonidesignal in tobacco cells. Plant Physiology and Biochemistry34, 399–408.

Maxwell DP, Lumsden RD, 1970. Oxalic acid production bySclerotinia sclerotiorum in infected bean and in culture.Phytopathology 60, 1395–8.

Movahedi S, Heale JB, 1990. The roles of aspartic proteinaseand endo-pectin lyase enzymes in the primary stages ofinfection and pathogenesis of various host tissues by differ-ent isolates of Botrytis cinerea Pers ex. Pers. Physiologicaland Molecular Plant Pathology 36, 303–24.

Nelson N, 1944. A photometric adaptation of the Somogyimethod for determination of glucose. Journal of BiologicalChemistry 153, 375–80.

Olsson K, 1989. Relationship between pectolytic enzymeactivity and rot development in potato tubers inoculatedwith Fusarium solani var. coeruleum. Journal of Phyto-pathology 124, 225–35.

Ried JL, Collmer A, 1985. Activity stain for rapid characteri-zation of pectic enzymes in isoelectric focusing and sodiumdodecyl sulfate-polyacrylamide gels. Applied and Environ-mental Microbiology 50, 615–22.

Rouet-Mayer MA, Mathieu Y, Cazale AC, Guern J, LauriereC, 1997. Extracellular alkalinization and oxidative burstinduced by fungal pectin lyase in tobacco cells are not dueto the perception of oligogalacturonide fragments. PlantPhysiology and Biochemistry 35, 321–30.

Walton JD, 1994. Deconstructing the cell wall. Plant Physiol-ogy 104, 1113–8.

Wattad C, Dinoor A, Prusky D, 1994. Purification of pectatelyase produced by Colletotrichum gloeosporioides and itsinhibition by epicatechin: a possible factor involved in theresistance of unripe avocado fruits to anthracnose. Molecu-lar Plant–Microbe Interactions 7, 293–7.

Wattad C, Freeman S, Dinoor A, Prusky D, 1995. A non-pathogenic mutant of Colletotrichum magna is deficient inextracellular secretion of pectate lyase. Molecular Plant–Microbe Interactions 8, 621–6.

Wijesundera RLC, Bailey JA, Byrde RJW, 1984. Production ofpectin lyase by Colletotrichum lindemuthianum in cultureand in infected bean (Phaseolus vulgaris) tissue. Journal ofGeneral Microbiology 130, 285–90.

Wijesundera RLC, Bailey JA, Byrde RJW, Fielding AH, 1989.Cell wall degrading enzymes of Colletotrichum linde-muthianum: their role in the development of bean anthrac-nose. Physiological and Molecular Plant Pathology 34,403–13.

Zucker M, Hankin L, 1970. Regulation of pectate lyasesynthesis in Pseudomonas fluorescens and Erwinia caroto-vora. Journal of Bacteriology 104, 13–8.

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