J Sci Food Agric 1996,72,86-90

A Chitinase and a ~-1,3-Glucanase Isolated from the Seeds of Cowpea (Vigna unguiculata L Walp) Inhibit the Growth of Fungi and Insect Pests of the Seed Valdirene M Gomes, Anthia Elenir A Oliveira and Jose Xavier-Filho* Departamento de Bioquimica e Biologia Molecular, Universidade Federal do Ceara, Caixa Postal 1065, 60001-970 Fortaleza, Ceara, Brazil (Received 21 November 1995; revised version received 19 February 1996; accepted 12 April 1996)

Abstract: A chitinase (22 000 Da) and a p-1,3-glucanase (26 000 Da) were isolated from cowpea (Vigna unguiculata) seeds and shown to deter development, in an in uitro assay, of the phytopathogenic fungi Colletotrichum lindemuthianum (agent of anthracnose, a disease of many plants) and Colletotrichum musae (agent of banana peel anthracnose). The isolated chitinase was also shown to negatively affect the development of the cowpea weevil (Callosobruchus maculatus) in an artificial seed system. These results suggest that the cowpea seed contains defence proteins that could be, if their levels are properly managed, utilised to promote increased protection of the plant towards attacking fungi and insects.

Key words : chitinase, B-1,3-glucanase, antifungal proteins, insecticidal proteins, defence proteins, cowpea seeds, Vigna unguiculata, Callosobruchus maculatus, Colletotrichum lindernuthianum, Colletotrichum musae.

INTRODUCTION specific pests and pathogens as a result of natural selec- tion or breeding (Gatehouse et a1 1990; Macedo et al

Cowpea (Vigna unguiculata) is a legume of West African 1993). origin whose seeds are rich (about 25%) in protein Many seed specific proteins which are thought to be (Bressani 1985) and which are widely consumed by poor components of the arsenal of defensive compounds uti- populations throughout the tropics (Singh and Rachie lised by plants are similar to some of the so-called 1985). The plant is considered to have a low level of pathogenesis related proteins (PR proteins) (Leah et al defences against herbivores and microorganisms in 1991 ; Huynh et al 1992). These proteins, mostly isolated general and this is thought to be the reason why the from leaves and whose synthesis is induced by signals culture shows very low yields (Latunde-Dada 1990). originating from pathogen attack or environmentally Nevertheless, several cowpea varieties or cultivars are produced, belong to several classes, among them are the able to display defences against many viruses, bacteria, hydrolases, chitinases and /?-1,3-glucanases. These fungi and insects (Singh and Singh 1992). Many of these enzymes are thought to deter, in uiuo, the advance of defences, constitutive or induced ones, are of protein fungal attack in incompatible plant-fungal interactions nature, like the trypsin inhibitors (Xavier-Filho and (Roberts and Selitrenikoff 1988; Arlorio et al 1992; Ventura 1988) and are commonly found in all plant Ignatius et al 1994). seeds in general. These are directed either towards The role of chitinases and /?-1,3-glucanases during deterring general predators or are targeted towards host infection of seeds by fungi, or attack by seed eating

insects, if any, is not well understood. One reason for * To whom correspondence should be addressed at: Labor- this lack of is the low levels of these atbrio de Quimica e Fun@o de Proteinas e Peptideos, Centro enzymes found in these organs. de Biocitncias e Biotecnologia, Universidade Estadual do Norte Fluminense, Av. Alberto Lamego, 2000-28015-620 The results presented in this paper suggest that Campos dos Goytacazes, Rio de Janeiro, Brazil. cowpea seeds contain chitinases and j-1,3-glucanases

J Sci Food Agric 0022-5142/96/$09.00 0 1996 SCI. Printed in Great Britain 86

Cowpea seeds chitinase and /3-glucanase 87

and that these, if their levels are adequately managed, can be utilised to promote increased protection of the plant towards attacking fungi and insects.

MATERIALS AND METHODS

Plant material

Seeds of cowpea (Vigna unguiculata (L) Walp) used in this work were of the cultivar CE-31 (Pitiuba) and were provided by the Centro de CiCncias Agrarias, Uni- versidade Federal do Ceara, Fortaleza, Brazil.

Purification of enzymes

A finely divided meal (60 mesh) was obtained by grind- ing the decorticated seeds in a coffee mill. Proteins were extracted (1 : 5 meal to buffer ratio) with 0.1 M Tris-HC1 buffer of pH 7.5 for 1 h at room temperature and cen- trifuging for 20 min at 7500 x g . The supernatant obtained was subsequently fractionated by ammonium sulfate precipitation in three fractions, corresponding to 30, 60 and 90% saturation, all containing chitinase and /3-glucanase activities.

DEAE-Sepharose (equilibrated with 0.01 M Tris-HC1 buffer, pH 8) was utilised for the separation of the activ- ities. Five hundred milligrams of the 30/60 ammonium sulfate fraction, dissolved in the same buffer was equili- brated with the adsorbent and batch elution was ini- tially done by the equilibrium buffer, when we obtained fraction DI, followed by a solution of 1 M NaCl which furnished fraction DII. Both fractions were recovered by dialysis and lyophylisation.

Chitinase was purified by an affinity procedure employing chitin which was prepared according to the method of Hackman and Goldberg (1964) from pow- dered lobster shells. A column of chitin (3.5 x 10 cm) was equilibrated with 0.08 M sodium acetate buffer (pH 4.5) and fraction DII (500 mg in 50 ml of equilibrium buffer) was applied to the column. The column was developed initially with the equilibrium buffer followed by 0-08 M sodium acetate buffer of pH 3.2 and by 100 mM HC1. Protein peaks (3.0 ml fractions) were col- lected and recovered after dialysis and lyophylisation. A Sephadex G-50 column (60 x 2.5 cm) was equilibrated with 0.005 M ammonium bicarbonate (pH 8.0) and cali- brated with several proteins of known molecular weight (bovine serum albumin, ovalbumin, soya bean trypsin inhibitor and ribonuclease). Fraction C4 (20 mg in 10 ml), which was retained in the chitin column and desorbed by 0-1 M HCl, was submitted to two chro- matographic cycles in Sephadex G-50.

For the purification of /3-1,3-glucanase we employed a smaller column ( 5 x 1.6 cm) of chitin which was equili- brated with 0.05 M phosphate buffer, pH 7-6. The

protein fraction which was not adsorbed by DEAE- cellulose at pH 8.0 (DI, see above) was dissolved (15 mg in 10 ml) in 0.05 M phosphate buffer, pH 7.6 and applied to the column. This was initially developed with equilibrium buffer followed by 100 mM HCl. Protein peaks (2.5 ml fractions) were collected and recovered after dialysis and lyophylisation. The protein fractions which were not absorbed by the chitin column at pH 7.6 and containing /3-1,3-glucanase activity were pooled and submitted to two cycles of gel filtration chromatog- raphy in Sephadex G-75 (column of 2.5 x 50 cm) and Sephadex G-50 (column of 1.8 cm x 35) both equili- brated and developed with 0.005 M ammonium bicar- bonate. A final ion-exchange chromatographic step in CM-Cellulose (column equilibrated with 0.08 M acetate buffer, pH 5.0) was employed to purify the enzyme. The fraction rich in /3-1,3-glucanase activity, obtained in the Sephadex G-50 chromatography, was applied ( 5 mg in 10 ml of equilibrium buffer) to the column and the chromatography developed with the 0.08 M acetate buffer, pH 5.0 followed by an NaCl gradient (0 to 0-5 M, 60 ml). Protein peaks (1.5 ml fractions) were collected and recovered after dialysis and lyophylisation.

Activity of enzymes

The activity of chitinase was determined by the method of Hackman and Goldberg (1964) which employs chitin azure (Sigma Chemicals Co, St Louis, MO, USA) as substrate. Five milligrams of this coloured chitin deriv- ative was incubated with a solution of protein fractions containing chitinase activity in a final volume of 3.0 ml in 0.08 M sodium acetate buffer, pH 4.5. After incu- bation at 37°C for 48 h, with continuous stirring the supernatant was recovered by centrifugation (2000 x g, 5 min) and the absorbance at 575 nm read. One unit of activity was defined as the concentration of chitinase which produces an absorbance difference of 0.001.

/3-1,3-Glucanase was assayed by the method of Fink et aZ(l988) which employs laminarin (Sigma Chemicals Co, St Louis, MO, USA) as substrate. Enzyme- containing solutions (0.2 mg in 0.1 ml) in 0.01 M Tris- HCl buffer, pH 8.0 were incubated at 37T, for 6 h, with substrate (0.25 ml of a 2 mg ml-' laminarin solution in 0-05 M acetate buffer, pH 5.0) in a final volume of 0.5 ml. Colour was developed by the copper- arsenomolibdate method (Nelson 1944; Somogyi 1952) and the absorbance was read at 500 nm. One unit of activity was defined as the concentration of enzyme which produces an absorbance difference of 0.001.

Fungi

The following phytopathogenic fungi, obtained from the Phytopathology Section of the Centro de CiCncias Agrarias (Universidade Federal do Ceara, Fortaleza,

88 V M Gomes et a1

Brazil), were utilised for testing growth inhibiting properties of isolated fractions: Colletotrichum lindemu- thianum (an agent of anthracnose, a disease of many plants), Colletotrichum musae (an agent of banana peel anthracnose), Fusarium oxysporum (causing wilting of banana) and Macrophomina phaseolina (causing root and stem rot in cowpea).

Antifungal activity of the enzyme preparations was carried out under sterile conditions using the disc-plate diffusion assay as described by Roberts and Selitrenikoff (1988). Fungi were inoculated in the centre of Petri dishes (8.5 cm diameter) containing Sabouraud Dex- trose Agar. After incubation for 48 h at room tem- perature (25°C) to allow for spore germination and vegetative growth, sterile filter paper discs (Whatman 3MM) were radially distributed on the agar surface, close to expanding mycelial mat and 50 pl of the enzyme solutions (1% of S2 and P2’ fractions in 0.008 M acetate buffer, pH 4.5) were applied to the discs. The Petri dishes were then incubated for 24 h at 25°C. In this manner if the enzyme being tested is an antifungal protein a crescent-shaped zone of inhibition of fungal growth is observed around the disc.

Insects

Individuals from a colony of Callosobruchus maculatus (F) (Coleoptera : Bruchidae), the cowpea weevil (Jackai and Daoust 1986), maintained in the Departamento de Bioquimica e Biologia Molecular were used for feeding experiments. Artificial seeds (400 mg) made by pressing cowpea seed flour with additions (0.0, 0.1, 0.2, 0.5, 1.0, 2.0 and 4.0% of protein fraction) in a cylindrical brass mold with the help of a hand press were utilised for infestation by cowpea weevil females (Macedo et at 1993). After infestation and eclosion, two seeds with three eggs each were left for insect development in glass tubes (20 x 2 cm) covered with cotton plugs and placed in growth chamber (25°C and 80% RH). After 20 days the seeds were broken apart and the number and weight of surviving larvae were recorded. Due to restrictions on the amount of protein samples available we per- formed the experiment only once. Experimental points were utilised to fit dose-responses curves (quadratic correlation) from which we calculated the LD,,, and WD,,, for each protein fraction.

Polyacrylamide gel electrophoresis

SDS-polyacrylamide gel electrophoresis was performed according to the method of Laemmli (1970). Non- denaturing polyacrylamide gel electrophoresis was done by the method of Davis (1964). Proteins utilised as molecular weight standards for PAGE-SDS were bovine serum albumin (66 kDa), carbonic anhydrase

(29 kDa), trypsinogen (24 kDa) and a-lactoglobulin (14.2 kDa).

Protein determination

Protein was determined by the dye-binding method of Bradford (1976) or by the absorbance at 280 nm.

RESULTS AND DISCUSSION

The purification of both a chitinase and a /I-glucanase from seeds of cowpea (Vigna unguiculata) was achieved through the utilisation of ion-exchange, chitin affinity and size exclusion chromatographies. DEAE-Sepharose was initially utilised for the separation of basic and acidic proteins contained in the 30/60 ammonium sulfate fraction. The adsorbed fraction (DII) from the first ion-exchange step was further fractionated by chitin affmity chromatography and the more strongly bound fraction (C4 pool) was submitted to gel-filtration in Sephadex G-50. Chitinase rich fractions from this step were re-chromatographed in the same column after which we obtained a highly purified (ST, 81.3-fold purification) chitinase preparation with an yield of 2.1% (Table 1).

Non-adsorbed fractions (DI) from the ion-exchanger step were fractionated by chitin affinity chromatog- raphy (pH 7.6) and the break-through peak (GI) was submitted to gel-filtration in Sephadex G-50. After rech- romatography /I-glucanase rich fractions were finally purified employing a cation exchanger step. The prep- aration obtained (CM 2’) was 22.6 fold enriched and represented 0.74% of the initial crude extract (Table 1). Low yields of both enzymes can be explained by the distribution of activities in several fractions from the ammonium sulfate, the chitin affinity step through the final ion-exchange chromatography (Table 1).

Polyacrylamide gel electrophoresis of the isolated 8- glucanase and chitinase enzymes gave single bands on the gel and indicated molecular masses of 26000 and 22000 Da, respectively (Fig 1). The value for the chitin- ase lies in the range of molecular weights found by several authors for chitinases from seeds (Awade et a1 1989; Leah et al 1991 ; Zhe-fu et af 1992). The molecular weight for the cowpea /I-glucanase reported here is in agreement with values reported previously for other plant materials (Wong and Maclachlan 1980; Awade et a1 1989).

There is a great interest in the elucidation of the role of both chitinases and /I-glucanases in plants since sub- strates for these enzymes are not commonly reported as being present in vegetable tissues. Most of the work on these hydrolases is based on the assumption that they are involved in defence mechanisms of plants against fungi. These hydrolases would act on the tips of growing fungal hyphae when newly deposited chitin

TABLE 1 Purification table for cowpea seeds chitinase and /3-1,3-glucanase

Fraction Chitinase /3-1,3-Glucanase

Protein Specific Yield PuriJcation Protein Specific Yield (YO) Purification (mg) activity (%) (fold) (mg) actioity (fold)

Crude extract

Fraction 30160 DI DII C4 s 2 S2’ P2‘ CM2

24320 1.41 100

6695 4.7 91.1 720 6.7 14.0

2260 14.6 96.0 28 42.9 3.5 9 101.1 2.6 6.3 114.6 2.1

1

3.3 4.7

10.3 30.4 71.7 81.3

24320

6695 720

2260 -

-

30 8

3.81

6.2 21.0 4.0

-

38.8 86.2

100

44.6 16.3 9.8

-

1.25 0.74

1

1.6 5.5 1 .o

- 10.2 22.6

would be accessible to chitinases secreted by the attacked plant (Chrispeels and Raikhel 1991). We tested both chitinase (S2) and j?-glucanase (P2’), at 1% concen- tration, for the growth inhibition of phytopathogenic fungi, namely C lindemuthianum (agent of anthracnose, a disease of many plants), C musae (agent of banana peel anthracnose), F oxysporum (causing wilting of banana) and M phaseolina (causing root and stem rot in cowpea). We observed inhibition of the growth of both C musae (Fig 2) and C lindemuthianum (not shown) sug- gesting a specific action of the cowpea chitinase and b- glucanase since no growth inhibition of the other utilised fungi was observed.

The action of the isolated chitinase on the larval development of the bruchid beetle C maculatus, which is

a pest of cowpea seeds throughout the tropics (Jackai and Daoust 1986), was measured utilising an artificial seed system. From the dose-response curves we calcu- lated a value of 1.5% for WD,,, ( I = -0.920; P > 0.01) and an LD50, > 4 (Y = -0.873; P > 0.01). This low value for WD,,, suggests that the isolated chitinase from cowpea affects development although it does not affect much the survival of the larvae of this insect (a high value for LD,,,). This toxic effect can be

Fig 2. Agar gel plate showing growth inhibition of the fungus Colletotrichum rnusae by (1) cowpea seed chitinase; (2) /3-1,3- glucanase; (3) chitinase plus /3-1,3-glucanase and (4) phosphate buffer. Colletotrichurn musae was inoculated in the centre of Petri dishes (8.5 cm diameter) containing Saboraud Dextrose Agar and incubated for 48 h at 25°C. After this incubation period filter paper discs imbibed in the test solutions YO solutions of the enzymes made up in 0.08 M, pH 4.5 acetate buffer) were distributed on the culture medium, in front of the developing fungi. The dishes were incubated at the same con-

Fig 1. Polyacrylamide gel electrophoresis of the purified /3-1, 3-glucanase and chitinase isolated from cowpea seeds. M refers to molecular weight markers, G and C correspond to

/3-1,3-glucanase and chitinase, respectively. ditions.

90 V M Gomes et a1

tentatively explained by the action of the enzyme on the chitin component of the peritrophic membrane in the midgut of insects (Richards and Richards 1977).

Not very much is known about the role of chitinases and glucanases in seeds but the absence of the sub- strates for these enzymes in the seeds and their presence as normal constituents of fungal cell walls and of the lining of insect midguts (in the case of the chitinases) strongly suggests a protective role for these hydrolases. Their presence, in cowpea seeds, together with sub- stances like cyanogenetic glucosides (Jaffe 1950), tannins and lectins (Xavier-Filho et af 1989), proteinase inhibi- tors (Xavier-Filho and Ventura 1988; Fernandes et a1 1991), variant vicilins (Macedo et al 1993) and many others may indicate that seeds, in general, are endowed with chemical defences against herbivores. Specificity of these defenses towards the several attacking pests and pathogens or their low levels in seeds could be some of the reasons why cowpea shows very low yields (Latunde-Dada 1990). Proper breeding techniques or genetic manipulations of the plant could endow the cowpea with adequate levels of defenses and lead to increased yields of this staple food in regions of the world where protein deficiency is very high.

ACKNOWLEDGEMENTS

The research was financed by the Brazilian agencies FNDCT/FINEP, CNPq and CAPES. The authors thank D r Katia V Sales Fernandes for critically reading this manuscript.

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