Mycol. Res. 97 (4): 461-466 (1993) Printed in Great Britain 461

Pectin degrading enzymes secreted by six isolates of Fusariumoxysporum

NURIA FERNANDEZ, BELEN PATINO AND COVADONGA VAZQUEZ

Departamento de Microbiologz'a III, Facultad de Biologz'a, Universidad Complutense, 28040-Madrid

The in vitro production of extracellular pectic activities by six isolates of Fusarium oxysporum was studied.A crude extracellular enzymic preparation can be produced from F. oxysporum with high pectic activity. F. oxysporum f. sp.

lycopersici (FOL race 1) accumulated the highest levels of endo-PG activity. Other strains, F. oxysporum f. sp. lycopersici (FOL race 2)

and F. oxysporum f. sp. radicis-lycopersici (FORL) reached about 20% of this activity. The PG of FOL (20) has been characterized asexo-PG, whereas all other vascular wilt pathogens produce endo-PG.

FOL strains exhibited a very similar pattern of major PG-activity in electrofocusing independent of races. One major band ofactivity had a pI of 7'0, and the other activity bands had pIs of 6'5, 6'15 and 5'9.

One of the two FORL isolates shared the patterns of vascular wilt, the other presented a characteristic band with a pI of 7'45.

Fusarium oxysporum Schlecht. f. sp. lycopersici (Sacc.) Snyder &

Hansen is the organism which causes vascular wilt in tomatowith two or three pathogenic races (Gerdeman & Finley,1951; Gabe, 1975; Armstrong & Armstrong, 1980). In Spaintwo races have been described. Race 2 was described in Spainin 1978 (Tello & Perez, 1978).

F. oxysporum f. sp. radicis-lycopersici Jarvis & ShoemakerGarvis & Shoemaker, 1978) was first detected on tomato in1988 (Tello & Lacasa, 1988). It is responsible for foot, crownand root rot in this cultivar. The symptoms it causes aredifferent from those caused by vascular wilt fusaria, and it isbeginning to be a serious problem in Spain.

For Bosland (1988) the concept of species in F. oxysporumis morphological because it lacks the teleomorphic state andmoreover phenotypic variability is extensively recognized,thereby making this problem very complex.

On the other hand, the presence of subspecific formsdesignated as formae speciales and races is based on hostspecificity and pathogenicity. Also it is necessary to considerthe presence of diverse parasitic abilities in one particular race.

Pathogenicity tests are the only criteria for differentiatingthe forms of F. oxysporum. This is a laborious task and isalways influenced by a multitude of variables (Armstrong &Armstrong, 1978).

F. oxysporum has the ability to release a battery ofextracellular enzymes to degrade pectin and cellulose, themajor plant primary cell wall components. Degradation ofpectin involves breakdown of polygalacturonic acid throughtwo enzymatic processes: transeliminative and hydrolytiCreactions.

Pectic enzymes of pathogens can contribute to thedevelopment of wilt and root rot diseases (Dimond, 1970;

Cooper & Wood, 1980; Pegg, 1981) in spite of this ideahaving been questioned on other occasions (Howell, 1976).

In this work the pectic enzyme patterns in six isolates of F.oxysporum from different races and pathotypes have beenstudied with the object of contributing to the knowledge ofthe relationship between pectic enzymes and various parasiticabilities.

MATERIALS AND METHODS

Organism and culture conditions

Isolates of races 1 and 2 of F. oxysporum f. sp. lycopersicireferred to as FOL-1A FOL-IB and FOL-2C, FOL-2Drespectively and two isolates of F. oxysporum f. sp. radicis­lycopersici, referred to as FORL-E and FORL-F, were providedby Dr Tello (LN.LA., Madrid) from diseased tomato cropsgrown on the Spanish Mediterranean coast. The fungi weremaintained as stock cultures on potato-dextrose-agar (PDA)slants at 4 0c.

Modified Czapek-Dox medium (Vazquez et ai., 1986) with1 % pectin was used to grow the fungus in static cultures at26° in 100 ml Erlenmeyer flasks containing 25 ml medium.Periodically samples were taken from two replicate flasks.

Mycelium was separated from culture fluid by centrifugation(5000 g). The culture fluid was utilized as a source of enzymes.

Enzyme assays

Polygalacturonase activity (PG) (EC 3.2. 1 . 82) was assayedby following the release of reducing groups from 0'1 % pectin(from apple, Fluka) or polygalacturonic acid in 50 mM sodium

Polygalacturonases in Fusarium oxysporum 462

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Incubation time (days)Fig. 1. Variation of pH (a), mycelial dry weight (b), and protein (c) with incubation time. F. orysporum f. sp. lycopersici FOL race lA(e), FOL race IB (0), FOL race 2C (0), FOL race 2D (.l, and Fusarium orysporum f. sp. radicis lycopersici FORL E (~) and FORL F

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acetate buffer pH 5'2 according to the methods of Somogyi(1945) and Nelson (1944). One unit of enzymic activity wasdefined as the amount releasing I 1lI110l of gaIacturonic acid inI min. EndopolygaIacturonase (Endo-PG) (EC 3.2. I. 15) was

determined by viscometry, viscosity measurements of reactionmixtures containing 2 ml of enzyme and 8 ml I % pectin in50 mM sodium acetate buffer pH 5·2 in Cannon-Fenskeviscometers (300) at 25°.

N. Fernandez, B. Patino and C. Vazquez

This activity was expressed as relative viscometric units(RVU), and these units were defined as 1000/t50, where t50is the time taken for 50 % viscosity reduction.

Pectinlyase (PL) (EC 4.2.2.10) was assayed by an increasein absorption at 235 nm of 0'1 % polygalacturonic acid in50 mM sodium acetate buffer pH 5'2. One unit of PL activitywas defined as the amount of enzyme which produced 1 ~olof unsaturated products in one min, based on the molarextinction coefficient (4600) of unsaturated compounds.

Enzyme and substrate controls were carried out.Protein concentration was determined by the Lowry method

(Lowry et a1., 1951) using bovine serum albumin as a standard.

Analysis of reaction products

Pectolytic fragments produced by the action of PC on pectinwere analysed by cellulose chromatography (Merck). Theenzymes were incubated at 30° with 0'1 % pectin in 50 mMsodium acetate buffer pH 5'2, for different periods. Afterincubation, samples (20 IJI) were analysed. The chromatogramswere developed for 8 h with an n-butanol-water-acetic acid(5:3:2, v/v) solvent system and dried. Pectic fragments werevisualized in 0'05 % bromophenol blue, 1 mM Tris hyd­rochloride pH 7'5, 95 % ethanol. Areas containing fragmentsappeared yellow on a blue background.

Analytical electrophoresis

The culture filtrates were concentrated by ultrafiltration(Amicon) before application to electrophoresis or isoelectricfocusing to detect any differences in the isoenzyme andprotein patterns.

The electrophoretic method of Cruickshank (Cruickshank &Wade, 1980) was used. Culture fluids were added to the 1%pectin polyacrylamide gels. After electrophoresis, gels wereincubated in 0'1 Mmalic acid at 25° for 5-30 min, and stainedovernight in 0'01 % ruthenium red.

Isoelectric focusing (IEF) was carried out on ultrathin(0'4 mm) 5% polyacrylamide gels, with 2% Bio-Lyte 5/8ampholytes (Bio-Rad). Cels were pre-electrofocused at 125 Vfor 15 min, and then 13 IJI of concentrated extracellularsupernatant from each isolate were applied to the gel.Subsequent electrofocusing was at 100 V for 15 min; 200 Vfor 15 min and 450 V for 60 min. After electrofocusing, thegels were washed in suitable buffer and incubated in a 1%pectin in 50 mM sodium acetate buffer pH 5'2 for 60 min andstained overnight in 0'02 % ruthenium red solution.

After staining, the gels were washed in distilled water untilthe bands became evident.

463

highest accumulation occurring between the seventh andtenth days in FOL (race 1 and 2), whereas only low PCactivity, occurred with the two isolates of FORL. The graphshows a rapid decline in PC after the tenth day. No PL waspresent in filtrates from pectin cultures of all six isolates duringthis period of time. These two enzymes rarely appeartogether; one reason could be the influence of culture pH(Cooper & Wood, 1980).

Endo-PC activity was detected during the seventh and thetenth day of culture, FOL race-1A having accumulated thehighest levels, with 384'6 rvu at 10 days. The other isolatesaccumulated only about 20% of this activity, as is usual forthis pathogen (Cooper et a1., 1981) (Table 1).

It is remarkable that in spite of greater aggressiveness byFOL race 2, the ability to produce enzymes under theseconditions was lower than that of FOL race 1.

The resolution of the action pattern of the hydrolytic pecticenzymes (endo-PC or exo-PC) was determined by analysingreaction products by thin-layer chromatography.

All the FOL race 1 strains and one of the FOL race 2 strainsgenerated oligomers and monomer, indicating that thepolymer was cleaved in an endo-fashion. The sole reactionproduct was never a monomer.

Strain FOL 2D cleaved the polymer in an exo-fashion,because a single reaction product was released from thebeginning of the incubation. This product accumulated duringthe course of incubation, but no high-molecular weightproducts were detected.

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Incubation time (days)

Fig. 2. Time-course of polygalacturonase activity produced byFusarium oxyspornm f. sp. lycopersici FOL race lA (el. FOL race IB(Ol, FOL race 2C (Ol, FOL face 2D (.), and Fusarium oxyspornrnf. sp. radicis-lycopersici FORL E (.0.) and FORL F CAl.

Table 1. Endo-PG activity of isolates of Fusariumoxysporum

Endo-PG activity (RVUj'

RESULTS AND DISCUSSIONIsolate 7th day 10th day

The time-course of changes in pH, mycelial dry weight, andprotein content of culture filtrate (Fig. 1) were followed over14 days of incubation.

Figure 2 shows the production over a 14-day period of PCby six isolates in the presence of pectin as a carbon source. PCwas detected in fluids from cultures on the third day, the

FOL lA 357·1FOL IB 61·6FOL 2C 71"4FOL 20 79·4FORL E 37'6FORL F 11"1

• RVU = Relative Viscometric Units.

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Polygalacturonases in Fusarium oxysporum 464

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Fig. 3. Analysis of the reaction products of the extracellular hydrolytic pectin enzymes produced by strains of Fusarium oxysporum f. sp.lycopersici (lA 1B, 2C, and 2D) and F. oxysporum f. sp. radicis-lycopersici (E and F), incubated with apple pectin.

N. Fermindez, B. Patino and C. Vazquez

FORL isolate F

FORL isolate E

FOL isolate D

FOL isolate C

FOL isolate B

FOL isolate A

Fig. 4. Localization of polygalacturonase activity in polyacrylamidegel electrophoresis from culture filtrates of F. oxysporum f. sp.lycopersici and F. oxysporum f. sp. radicis lycopersici.

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FORL-E released oligomers and monomer. The strainFORL F accumulated monomer after 5 h of incubation whichcorresponds with a major exo-PC (Fig. 3).

When the culture filtrates were run on pectin acrylamidegels, one PC activity band was obtained for FOr. (race 1 and2) and FORL (E isolate). These activities were visible withinonly five minutes of incubation in malic acid. FORL (F isolate)shows two PC activity bands, one being a slower movingband (Fig. 4).

When subjected to isoelectric focusing these proteins wereresolved into several bands widely distributed between pI 5'9and 7'45 (Fig. 5). The proteins from different vascular isolateswere indistinguishable or showed little differences alone inintensity of staining. However, the proteins from FORL (Fisolate) differed in number and pI.

30

465

Strains FOr. (races 1 and 2) exhibited a very similar patternof major PC activities in electrofocusing of supernatantcultures. One major band of activity had a pI of 7'0, and theother three activity bands had a pI of about 6'5,6'15 and 5'9.In the case of the two FORL isolates, one of them shared theapproximate patterns of the vascular fungi. .The other FORLstrain (F-isolate) presented one characteristic and principalband with pI of 7'45, and two minor bands with pI 7'0 and6'5, slightly more basic than those reported by other authors(Martinez et aI., 1991).

Attempts to correlate enzyme production in vUro by fungito pathogenicity in vivo frequently present contradictory orinconclusive results, probably because the differential pro­duction of enzymes by organisms in culture may be unrelatedto their activities in the host plant or because there are otherfactors involved.

Comparison of enzyme production by FOL and FORLstrains indicates that vascular wilt fusaria were the betterpectic enzyme producers, the two FOL races producing thesame patterns of pectic enzymes. Solely quantitative differ­ences were noted and the greatest activities were not alwayssystematically correlated with major virulence. The races arenot clearly defined and there has been difficulty in theirdifferentiation (Armstrong & Armstrong, 1978).

PACE and IEF application to the identification of Fusariumpathotypes has, in fact, been a rapid method in thedifferentiation or clarification of pathogenic races, and it doespermit the detection of important differences in several PCactivities for some pathotypes which would otherwise beindistinguishable based solely on their morphology andcolony characteristics in pure culture.

We thank Mrs M. Wood for helpful improvement of theEnglish manuscript.

This work was supported by Comunidad Autonoma deMadrid (CAM). Programa de Investigaci6n Agroalimentaria.CI88/90.

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Armstrong, G. M. & Armstrong, J. K. (1980). Formae speciales and races ofFusarium oxysporum causing wilt diseases. In Fusarium, Diseases, Biology andTaxonomy (ed. P. E. Nelson, T. A. Toussoun & R. J. Cook), pp. 391-399.

Pennsylvania State University Press: University Park and London.Bosland, P. W. (1988). Fusarium oxysporum, a pathogen of many plant species.

Advances in Plant Pathology 6, 281-289.

Cooper, R. M., Wardman, P. A. & Skelton, J. E. M. (1981). The influence ofcell walls from host and non-host plants on the produdion and adivity ofpolygaladuronide-degrading enzymes from fungal pathogens. PhysiologicalPlant Pathology 18, 239-255.

Cooper, R. M. & Wood, R. K. S. (1980). Cell wall-degrading enzymes ofvascular wilt fungi. III. Possible involvement of endo-pedin lyase inVerticillium wilt of tomato. Physiological Plant Pathology 16, 285-300.

Cruikshank, R. H. & Wade, G. C. (1980). Detedion of pedic enzymes inpedin-acrylamide gels. Analytical Biochemistry 107, 177-181.

Dimond, A. E. (1970). Biophysics and biochemistry of the vascular wiltsyndrome. Annual Review of Phytopathology 8, 301-322.

Gabe, H. L. (1975). Standardization of nomenclature for pathogenic races of

MYC 97

Polygalacturonases in Fusarium oxysporum

Fusarium oxysporum f. sp. lycopersici. Transactions of the British MycologicalSociety 42, 156-159.

Gerdeman, J. W. & Finley, A. M. (1951). The pathogenicity of races 1 and 2of Fusarium oxysporum f. sp. lycopersici. Phytopathology 41, 238-244.

Howell, C. R. (1976). Use of enzyme deficient mutants of Verticillium dahliaeto assess the importance of pedolytic enzymes in symptoms of Verticilliumwilt of cotton. Physiological Plant Pathology 9, 279-283.

Jarvis, W. R. & Shoemaker, R. A. (1978). Taxonomic status of Fusariumoxysporum causing foot and root rot of tomato. Phytopathology 68,1679-1680.

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Martinez, M. L Alconada, M. T., Guillen, F., Vazquez, c. & Reyes, F. (1991).Pedic adivities from Fusarium oxysporum f. sp. melonis. FEMS MicrobiologyLetters 81, 145-150.

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Nelson, H. (1944). A photometric adaptation of the Somogyi method fordetermination of glucose. Journal of Biological Chemistry 153, 375-380.

Pegg, G. F. (1981). Biochemistry and physiology of pathogenesis. In FungalWilt Diseases of Plants (ed. M. E. Mace, A. A. Bell & C. H. Beckman),pp. 193-253. Academic Press: New York.

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Tello, J. c. & Lacasa, A. (1988). La podredumbre del cuello y de las raices,causada por Fusarium oxysporum f. sp. radicis-Iycopersici, nueva enfermedaden los cultivos de tomate. Boletr'n de Sanidad Vegetal. Plagas 14, 307­312.

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