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Production of pectolytic enzymes by Curvularia senegalensis, a phytopathogenic fungus.

Abstract. -- The effect of different carbon sources on the production of extracellular pectinases of Curvularia senegalensis was investigated. Production of polygalacturonase and pectinesterase was induced by sodium polypectate, pectin and lactose. Production of these enzymes was repressed or completely inhibited in the control cultures containing maltose or glucose as carbon sources and enzyme inducers. Maximum production of polygalacturonase and pectinesterase was induced when sodium polypectate was used as carbon source. The results of this work indicate that the pectic enzymes of C. senegalensis may be produced in sequence, i.e., the production of polygalacturonase is followed by the secretion of pectinesterase.

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Direct penetration of susceptible hosts by the infective hyphae of phytopathogenic fungi is facilitated by the production of cutinases (Agrios 1988). Penetration of the host is followed by disintegration of the pectic substances of the middle lamella and the primary cell wall by pectolytic enzymes (Kenaga 1974; Agrios 1988). Production of pectolytic enzymes is induced in plant pathogenic fungi when these organisms are grown on media containing various carbon sources and enzyme inducers. These carbon sources include sugar polymers (Cooper & Wood 1973), sodium polypectate and pectin (Pegg 1981; Alana et al. 1989) and sugar beet cell walls (Bugbee 1990).

Curvularia senegalensis (Speg.) Subram., is primarily a pathogen of cereal crops and grasses including maize and sugar cane (Cook 1981; Nyvall 1989). The main objectives of this work were to study the components of extracellular cell wall degrading enzymes of C. senegalensis and to determine the effects of the carbon source on the production of these enzymes by the test fungus.

MATERIALS AND METHODS

Organism and culture conditions. -- Stock cultures of C. senegalensis were maintained on potato-dextrose-agar slants (PDA, Difco, B13). The fungus was previously grown in 250 mL flasks with 125 mL of a medium containing: 0.02% MgSO4.7H2O, 0.01% Ca(NO3)2.4H2O, 2.0% glucose in Na-citrate buffer at pH 4.8. After four days growth at 26[degrees]C, 5 mL of mycelium inoculum was washed twice in distilled water and transferred to the growing medium. The medium for the production of pectinases contained: 0.15% NH4NO3, 0.24% K2PO4, 0.08% MgSO4, 0.08% Ca(NO3)2.4H2O, 0.72 ppm Fe(NO3)3.9H2O, 0.44 ppm ZnSO4.7H2O, 2.0 ppm MnSO4.4H2O, 0.40 ppm ZnC12 and 1.0% carbohydrate. The carbohydrates used as carbon sources and enzyme inducers included: sodium polypectate, apple pectin, xylan (Sigma Chemical Company), lactose (Mallinckrodt Chemical Works) maltose and sucrose (Fisher Scientific). The control cultures had glucose (Fisher Scientific) as the sole carbon source. The pH of the growing medium was adjusted to 5.0 with 0.1 N KOH. Incubation of the cultures was carried out for seven days in covered 250 mL flasks on an orbital shaker at 80 rpm at 24[degrees]C.

Enzyme preparation and assays. -- Culture fluids were collected after seven days of growth in the liquid medium. The culture fluids were centrifuged (4,500 rpm, 30 minutes, 10[degrees]C) to obtain a clear supernatant. The supernatant was subsequently used for the determination of extracellular enzyme activity. For simplification, the collected supernatant is hereafter referred to as the enzyme.

Polygalacturonase (Pectinase, EC 3.2.1.15). -- Polygalacturonase activity was measured by combining 1 mL of enzyme with 10 milligrams of sodium polypectate in 1 mL of 0.05 M sodium citrate buffer (pH 5.0) and incubating the reaction mixture for 120 minutes at 40[degrees]C. The tubes were stirred once during incubation. After centrifugation, the concentration of galacturonic acid or its reducing sugar equivalent in the supernatant was determined by the dinitrosalicylic acid reagent of Miller (1959).

Pectinesterase (Pectin methylesterase, EC 3.1.1.11). -- Pectinesterase activity was measured by combining 1 mL of enzyme with 10 milligrams of pectin in 1 mL of 0.05 M sodium citrate buffer, (pH 5.0). The reaction mixture was incubated at 40[degrees]C for 120 minutes. After centrifugation, the concentration of galacturonic acid or its reducing sugar equivalent in the reaction mixture was measured by the dinitrosalicylic acid reagent of Miller (1959).

Protein determination. -- Extracellular total protein in the crude supernatant was determined with the BCA reagent (Pierce Chemical Company) using bovine serum albumin as standard.

Data analysis. -- All enzyme activity tests and protein determinations were replicated four times. Enzyme activities were expressed as units of specific enzyme activity (Usp) and represent means plus or minus the standard deviation of four replications. Each unit of enzyme specific activity was calculated as the amount of enzyme that liberated one micromole of galacturonic acid (or its reducing sugar equivalent) per minute per milligram of protein under the assay's conditions. Statistical analyses of experimental data were made with one-way analysis of variance (ANOVA) and Duncan's multiple range test (MRT).

RESULTS

Extracellular protein. -- The total extracellular protein that was measured in the fluids of the liquid cultures of this study was used to calculate the enzyme specific activities of the fungus grown in liquid media containing different carbon sources and enzyme inducers. Maximum concentration of extracellular protein (38.60 milligrams per mL) was measured in fluids collected from cultures containing sucrose as the carbon source (Table 1). The accumulation of protein in the fluids of cultures containing sucrose was significantly higher (P = 0.05) than the concentration of total protein measured in fluids containing other carbon sources. Total protein accumulated in the fluids of cultures containing pectin, lactose or glucose was significantly higher (P = 0.05) than the protein accumulated in cultures containing sodium polypectate, xylan or maltose (Table 1). The lowest amount of total protein (0.92 milligrams per mL) was measured in fluids collected from cultures containing sodium polypectate.

Polygalacturonase. -- Production of polygalacturonase was induced in cultures containing sodium polypectate, pectin, lactose and sucrose as carbon sources and enzyme inducers. No polygalacturonase activity was detected in the fluids obtained from cultures containing xylan, maltose or glucose as carbon sources (Table 1). Maximum polygalacturonase activity (21.38 Usp) was measured in fluids collected from cultures containing sodium polypectate (Table 1). This activity was significantly higher (P = 0.05) than the activities assessed in fluids obtained from cultures with pectin, lactose or sucrose (1.39, 1.40 and 0.60 Usp, respectively).

Polygalacturonase activities measured in fluids from cultures with pectin or lactose as carbon sources were significantly higher than the activity determined in fluids from cultures containing sucrose (Table 1). No significant difference was determined in the polygalacturonase activities measured in fluids of cultures containing pectin or lactose. The lowest polygalacturonase activities were measured in fluids containing sucrose.

Pectinesterase. -- Production of pectinesterase was induced in cultures containing sodium polypectate, pectin, xylan and lactose as carbon sources. Maximum pectinesterase specific activity (12.43 Usp) was measured in fluids harvested from cultures containing sodium polypectate (Table 1). This activity was significantly higher (P = 0.05) than the activities assessed in fluids obtained from cultures with pectin, xylan or lactose (0.27, 2.11 and 0.49 Usp, respectively). The pectinesterase activity measured in fluids obtained from cultures containing xylan (2.11 Usp) was higher than the activity measured in fluids collected from cultures with pectin or lactose (0.27 and 0.49 Usp, repectively) as carbon sources. However, the differences between these activities (1.84 and 1.62 Usp, repectively) were not significant. No pectinesterase activities were detected in fluids harvested from cultures containing maltose, sucrose or glucose as carbon sources and enzyme inducers.

DISCUSSION

Although the highest amount of extracellular protein determined in this study was found in fluids collected from cultures containing sucrose as the carbon source, it seems that only a small part of it was polygalacturonase. Whereas the production of polygalacturonase was induced in cultures of C. senegalensis containing sodium polypectate, pectin, lactose and sucrose as carbon source, sodium polypectate was the most effective inducer of this enzyme. Xylan, maltose or glucose did not induce the secretion of polygalacturonase by C. senegalensis under the conditions of this study.

It has been shown in similar studies that plant pathogenic fungi can be induced to secrete polygalacturonase and pectinesterase when grown in liquid media with the appropriate carbon source (Cleveland & McCormick 1987; Cooper & Wood 1973; Crawford & Kolattukudy 1987; De Lorenzo et al. 1987).

Pectinesterase activities were measured in fluids collected from cultures containing sodium polypectate, pectin, xylan and lactose. Production of both enzymes by the test fungus was repressed or completely inhibited when the growing medium contained maltose or glucose as the sole carbon source. Repression of the induction of polygalacturonase and other pectic enzymes by glucose or galacturonic acid used as carbon sources has been shown in other studies of phytopathogenic fungi (De Lorenzo et al. 1987; Leone & Van Den Heuvel 1987). The polygalacturonase accumulated in fluids obtained from cultures containing sodium polypectate was nearly two times higher than the amount of pectinesterase determined in the same fluids. In fluids containing pectin, the difference in enzyme activity was five times higher and in fluids with lactose as the carbon source, the activity of polygalacturonase was nearly three times higher than the activity of pectinesterase.

It is probable that the concentration of these enzymes in relation to each other is an indication of the sequence in which the enzymes were produced, i.e., the production of polygalacturonase is followed by the secretion of pectinesterase. The production in sequence of pectic enzymes has been demonstrated in similar studies of plant pathogenic fungi (Bahkali 1987; Leone & Van Den Heuvel 1987). In these studies, polygalacturonase was detected first in the culture filtrates. Pectinesterase appeared later.

SUMMARY

Secretion of polygalacturonase and pectinesterase was induced in liquid cultures of C. senegalensis when sodium polypectate, pectin or lactose were used as sole sources of carbon and enzyme inducers. Sodium polypectate was the most effective inducer in the production of both enzymes.
Table 1. Specific activities (1) of two pectolytic enzymes produced by
Curvularia senegalensis grown in liquid medium containing different
carbon sources.

 Enzymes
Carbon source Polygalacturonase Pectinesterase

Sodium polypectate 21.38 [+ or -] 1.01* 12.43 [+ or -] 5.30*
Pectin 1.39 [+ or -] 0.25 0.27 [+ or -] 0.10
Xylan 0.0 2.11 [+ or -] 0.28
Lactose 1.40 [+ or -] 0.66 0.49 [+ or -] 0.37
Maltose 0.0 0.0
Sucrose 0.60 [+ or -] 0.11 0.0
Glucose 0.0 0.0

 Enzymes
Carbon source Total protein (2)

Sodium polypectate 0.92 [+ or -] 0.32
Pectin 16.10 [+ or -] 0.65
Xylan 1.99 [+ or -] 0.15
Lactose 12.60 [+ or -] 0.07
Maltose 7.40 [+ or -] 0.26
Sucrose 38.60 [+ or -] 1.20*
Glucose 15.18 [+ or -] 0.75

(1) Usp/min/mg of protein. Mean [+ or -] SD of four replications.
(2) mg/mL.
* Using one-way ANOVA and Duncan's MRT, significantly different from
other values in the same group.


LITERATURE CITED

Agrios, G. N. 1988. Plant pathology. Academic Press, New York, Third ed., 803 pp.

Alana, A., A. Gabilondo, F. Fernando, M. D. Moragues, J. B. Dominguez, M. J. Llama & J. L. Serra. 1989. Pectin lyase production by a Penicillium italicum strain. Environ. Microbiol., 55:1612-1616.

Bahkali, A. H. 1987. Degradation of date palm (Phoenix dactylifera L.) cell walls by extracellular pectolytic enzymes produced by Verticillium albo atrum and Verticillium dahliae. Int. J. Trop. Plant Dis., 5:165-172.

Bugbee, W. M. 1990. Purification and characterization of pectin lyase from Rhizoctonia solani. Physiol. Mol. Plant Pathol., 36:15-26.

Cleveland, T. E. & S. P. McCormick. 1987. Identification of pectinases produced in cotton bolls infected with Aspergillus flavus. Phytopathology, 77:1498-1503.

Cook, A. A. 1981. Diseases of tropical and subtropical field, fiber and oil plants. MacMillan Publishing Co., New York, 450 pp.

Cooper, R. M. & R. K. S. Wood. 1973. Induction of synthesis of extracellular cell-wall degrading enzymes in vascular wilt fungi. Nature, 246:309-311.

Crawford, M. S. & P. E. Kolattukudy. 1987. Pectate lyase from Fusarium solani f. sp. pisi: Purification, characterization, in vitro translation of the messenger RNA, and involvement in pathogenicity. Arch. Biochem. Biophys., 258:196-205.

De Lorenzo, G., G. Salvi, L. Degra, R. D'Ovidio & F. Cervone. 1987. Induction of extracellular polygalacturonase and its messenger RNA in the phytopathogenic fungus Fusarium moniliforme. J. Gen. Microbiol., 133:3365-3374.

Kenaga, C. B. 1974. Principles of Plant Pathology. Waveland Press, Inc. Prospect Heights, Illinois. Second edition., 402 pp.

Leone, G. & J. Van Den Heuvel. 1987. Regulation by carbohydrates of the sequential in vitro production of pectic enzymes by Botrytis cinerea. Can. J. Bot., 65:2133-2141.

Miller, G. L. 1959. Use of dinitrosalicylic acid reagent for determination of reducing sugar. Anal. Chem., 31:426.

Nyvall, R. F. 1989. Field crop diseases handbook. Van Nostrand Reinhold, New York. Second edition, 817 pp.

Pegg, G. F. 1981. Biochemistry and physiology of pathogenesis. Pp. 193-253, in Fungal wilt diseases of plants (M. E. Mace, A. A. Bell and C. H. Beckman, eds.), Academic Press, New York, 640 pp.

Jacobo Ortega

Biology Department, The University of Texas-Pan American

Edinburg, Texas 78539

JO at: jortega@panam.edu
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Author:Ortega, Jacobo
Publication:The Texas Journal of Science
Date:Nov 1, 1998
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