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Cellulases of the phytopathogenic fungus Alternaria brassicae.

ABSTRACT. -- The extracellular cellulases of Alternaria brassicae and the effect of different carbon sources on the production of these enzymes were investigated. The production of endoglucanase was induced by all carbon sources tested. Results of this study suggest that production of endoglucanase and cellobiohydrolase by A. brassicae is constitutive. Key words: Alternaria; cellulases; enzymes.

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Many phytopathogenic fungi have the ability to penetrate directly the tissues of susceptible hosts. Penetration of the host by the infective hyphae of the fungus is facilitated by the production of cutinases (Agrios, 1988), followed by softening or distintegration of host tissues by cellulolytic and pectolytic enzymes produced by the pathogen (Kenaga 1974; Agrios, 1988). Production of these enzymes is induced in many plant pathogenic fungi when the these organisms are grown on media containing various sugar polymers (Cooper and Wood, 1973; Pegg, 1981; Ortega, 1990).

Gray leaf spot caused by Alternaria brassicae is a disease of cruciferous crops such as cabbage, broccoli, and cauliflower. These crops are cultivated twice a year in the Lower Rio Grande Valley of Southern Texas and represent an important part of the economy of that area. The main objectives of this work were to determine the components of extracellular cell wall degrading enzymes of A. brassicae 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 Alternaria brassicae were maintained on PDA slants (Difco, B13). The fungus was previously grown in 250-milliliter flasks with 125 milliliters of a medium containing: 0.02 percent MgS[O.sub.4]. 7[H.sub.2]0, 0.01 percent Ca(N[0.sub.3])[.sub.2]. 4[H.sub.2]0, 0.1 percent Peptone, 0.2 percent yeast extract, 2.0 percent glucose in Na-citrate buffer at pH 4.8. After four days growth at 26[degrees]C, five milliliters of mycelium inoculum was washed twice in distilled water and used to inoculate the cellulolytic growth medium. The medium for the production of cellulases contained: 0.15 percent N[H.sub.4]N[0.sub.3], 0.24 percent [K.sub.2]P[0.sub.4], 0.08 percent MgS[0.sub.4], 0.08 percent Ca(N[0.sub.3])[.sub.2]. 4[H.sub.2]0, Fe(N[0.sub.3])[.sub.3]. 9[H.sub.2]0. 0.72 ppm, ZnS[0.sub.4]. 7[H.sub.2]0, 0.44 ppm, ZnS[0.sub.4]. 7[H.sub.2]0, 0.44 ppm, MnS[0.sub.4]. 4[H.sub.2]0, 2.0 ppm, Zn[Cl.sub.2], 0.40, ppm, 0.1 percent Peptone, 0.1 percent yeast extract, 1.0 percent carbohydrate. The carbohydrates used as carbon sources and cellulase inducers were: sodium carboxymethyl cellulose (CMC, Hercules, Inc., type 7HF), microcrystalline cellulose (Sigma Chemical Co., type 50), and Xylan (Aldrich Chemical Company). The control cultures had glucose as the sole carbon source. The pH of the growing medium was adjusted to 4.8 with citric acid. Incubation of the cultures was carried out for seven days in covered 250-milliliters flasks on a orbital shaker at 100 rpm and 26[degrees]C. Enzyme Preparation and Assays.

Culture fluids were collected after seven days of growth by centrifugation (4500 rpm, 30 minutes, 10[degrees]C). The supernatant was subsequently used for the determination of extracellular activity. For simplification, the collected supernatant is hereafter referred to as the enzyme. All tests were replicated twice.

Endoglucanase (Endo-1, 4-B-glucanase, CMC-cellulase, EC 3.2.1.4). -- Endoglucanase activity was measured by a viscometric technique (Ortega and Baca, 1983). The viscometric data were then analyzed by a nonlinear method described by Ortega and Chance, (1988). The reaction mixture consisted of nine milliliters of 0.8 percent sodium carboxymethyl cellulose (Hercules Company) in 0.01 M potassium phosphate buffer (pH 4.5), and one milliliter of enzyme. Viscosity tests were made at 40[degrees]C.

Cellobiohydrolase (1,4-B-D-glucan cellobiohydrolase, EC 3.2.1.91). -- Extracellular cellobiohydrolase was determined with the method described by Tanaka et al. (1988). The substrate was a suspension of 50 milligrams per one milliliter of microcrystalline cellulose (type 50 by Sigma Chemical Company) in 0.05M sodium acetate buffer, pH 4.8. The reaction mixture consisted of nine milliliters of cellulose suspension and one milliliter of enzyme. The reaction tubes were incubated at 40[degrees]C for two hours. After centrifugation, the amount of total sugars in the supernatant was determined by the phenol-sulfuric acid method of Dubois et al. (1956).

Xylanase (EC 3.2.1.32). -- The activity of extracellular xylanase was determined as described by Saddler et al. (1985). The reaction mixture consisted of 10 milligrams of oat spelts xylan (Aldrich Chemical Company) in one milliliter of 0.05M sodium citrate buffer (pH 4.8) and one milliliter of enzyme.

Protein Determination

Total protein in the crude supernatants was determined with the BCA reagent (Pierce) using bovine serum albumin as standard. All enzyme activity tests as well as protein determinations were replicated twice. The results represent the average of two determinations. Enzyme activities were measured in units (U) of specific activity per milliliter (micrograms of substrate reacted per milliliter of enzyme per microgram of total protein).

RESULTS AND DISCUSSION

Endoglucanase

Maximum endoglucanase activity (1.95 U/ml) was measured in the culture fluids of A. brassicae when the fungus was grown in media containing microcrystalline cellulose as the carbon source and enzyme inducer (Table 1). This activity was 3.72 percent higher than the activity measured in the fluids obtained from the control cultures in which the carbon source was glucose (1.88 U/ml).

The endoglucanase activity measured in the culture fluids containing xylan (1.49 U/ml, Table 1) was 76.4 percent of that measured when the test fungus was grown in media with microcrystalline cellulose. The lowest endoglucanase activity (1.10 U/ml) was measured in cultures that had CMC as carbon source (Table 1). Most probably, the endoglucanase recorded in the control medium (1.88 U/ml) is a component of the complex of cellulolytic enzymes that is produced constitutively. It was found before (Ortega, 1990) that Fusarium oxysporum f. sp. lycopersici cultivated under the same conditions, produced constitutively a similar amount of endoglucanase. Cooper and Wood (1973) have shown that F. oxysporum f. sp. lycopersici and Verticillium albo-atrum grown with the proper enzyme inducers produce similar enzymes including galactosidase and polygalacturonase.

Cellobiohydrolase

Production of cellobiohydrolase was induced in those cultures containing microcrystalline cellulose, xylan and glucose. Maximum production of cellobiohydrolase (15.5 U/ml X [10.sup.-3]) was measured in fluids from cultures with microcrystalline cellulose as the carbon source (Table 1). When the test fungus was grown in media with xylan, the production of this enzyme was only 54.0 percent (8.38 U/ml X [10.sup.-3]) of that measured in the cultures with microcrystalline cellulose (Table 1). Cellobiohydrolase activity measured in the fluids from cultures with glucose was 61.9 percent (9.6 U/ml X [10.sup.-3]) of the activity measured in cultures with microcrystalline cellulose as the carbon source. Apparently, A. brassicae produces cellobiohydrolase in a constitutive manner. No cellobiohydrolase activity could be detected in cultures with CMC as the carbon source. It was found before (Ortega, 1990) that production of this enzyme is induced in cultures of F. oxysporuum f. sp. lycopersici with CMC as the carbon source.

Xylanase

Production of xylanase by the test fungus was induced in the cultures that had microcrystalline cellulose and xylan as the carbon source (Table 1). Maximum production of this enzyme (13.50 U/ml X [10.sup.-3]) was found in the cultures that had xylan. The production of xylanase measured in the cultures that had microcrystalline cellulose (10.90 U/ml X [10.sup.-3]) was 19.3 percent less than in the cultures that had xylan (Table 1). This enzyme was not found in the cultures that had CMC or glucose as carbon sources.

Apparently, neither CMC or glucose induces the production of xylanase by A. brassicae. It was shown before (Ortega, 1990) that CMC induces the production of xylanase by Fusarium oxysporum f. sp. lycopersici. Cooper and Wood (1973) used D-xylose to induce the production of xylanase in cultures of F. oxysporum f. sp. lycopersici and V. albo-atrum.
TABLE 1. Effect of different carbon sources on the production of
extracellular cellulolytic enzymes by Alternaria brassicae.

Carbon source Enzymes (1)
and cellulase Endo- Cellobio- Total
inducer glucanase (2) hydrolase (3) Xylanase (3) protein(4)

CMC 1.10 -- -- 960
Microcrystalline
 cellulose 1.95 15.50 10.90 220
Xylan 1.49 8.38 13.50 370
Control
 (glucose) 1.88 9.60 -- 500

(1) Specific activity, units/ml/ug of protein.
(2) Units/ml/ug of protein/minute.
(3) (Units/ml/ug of protein/hour) [10.sup.-3].
(4) Ug-micrograms/ml


LITERATURE CITED

Agrios, G. N. 1988. Plant pathology. Academic Press, New York, 3rd ed., 803 pp.

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

Dubois, M., K. A. Giles, J. K. Hamilton, P. A. Rebers, and F. Smith. 1956. Colorimetric method for the determination of sugars and related substances. Anal. Chem., 28:350-356.

Kenaga, C. B. 1974. Principles of plant pathology. Waveland Press, Inc. Prospect Heights, Illinois, 2nd ed., 402 pp.

Ortega, J. 1990. Production of extracellular cellulolytic enzymes by Fusarium oxysporum f. sp. lycopersici. Texas J. Sci., 42:405-409.

Ortega, J., and E. J. Baca. 1983. Viscometric measurement of the cellulase activity of a soil fungus. Texas J. Sci. 35: 261-267.

Ortega, J., and J. E. Chance. 1988. Determination of cellulase activities of fungi by nonlinear analysis of viscometric data. Texas J. Sci., 40: 323-329.

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.

Saddler, J. N., C. M. Hogan, and G. Louis-Seize. 1985. A comparison between the cellulase systems of Trichoderma harzianum E58 and T. reesei C30. Appl. Microbiol. Biotechnol., 22:139-145.

Tanaka, M., M. Idesada, R. Masuno, and A. O. Converse. 1988. Effect of pore size in substrate and diffusion of enzyme on hydrolysis of cellulosic materials with cellulases. Biotechnol. Bioeng., 32:698-706.

JACOBO ORTEGA

Biology Department, The University of Texas-Pan American, Edinburg, Texas 78539
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Author:Ortega, Jacobo
Publication:The Texas Journal of Science
Date:Aug 1, 1992
Words:1745
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