Insecticidal and antifungic proteins of the latex from Manihot glaziovii Muell. Arg.

Proteínas inseticidas e antifúngicas do látex de Manihot glaziovii Muell. Arg.

Insecticidal and antifungic proteins of the latex from Manihot glaziovii Muell. Arg.

LUCILENE S. PEREIRA¹, VALDIRENE M. GOMES², KÁTIA V. S. FERNANDES³, MAURÍCIO P. SALES⁴ and JOSÉ XAVIER-FILHO³

(recebido em 13/04/98; aceito em 18/09/98)

ABSTRACT - (Insecticidal and antifungal proteins of the latex from Manihot glaziovii Muell. Arg.). Analysis of the latex from Manihot glaziovii showed the presence of various enzymatic and inhibitory activities. The latex also presented an inhibitory effect on the development of cowpea weevil (Callosobruchus maculatus) in an artificial seed system and on the development, in an in vitro assay, of phytopathogenic fungi Colletotrichum gloesporioides, Fusarium solani and Macrophomina phaseolina. These results suggest the presence of substances, some of them of protein nature, involved in plant defense mechanisms in this exudation product.

RESUMO - (Proteínas inseticidas e antifúngicas do látex de Manihot glaziovii Muell. Arg.). Análise do látex de Manihot glaziovii mostrou a presença de várias atividades enzimáticas e inibitórias. O látex apresentou também um efeito inibitório sobre o desenvolvimento do gorgulho/caruncho do feijão-de-corda (Callosobruchus maculatus) em um sistema de sementes artificiais e sobre o desenvolvimento, em um ensaio in vitro, dos fungos fitopatogênicos Colletotrichum gloesporioides, Fusarium solani e Macrophomina phaseolina. Esses resultados sugerem a presença de substâncias, algumas delas de natureza protéica, envolvidas em mecanismos de defesa de plantas neste exsudato.

Key words - Latex, chitinase, b -1,3-glucanase, proteinase inhibitors, defense proteins

Introduction

Plants actively react to pathogen and insect attack by a complex of defense reactions that can be local or systemic, constitutive or inducible. Various classes of proteins have been implicated in resistance mechanisms of plants against pathogens and insects (Shewry & Lucas 1997). Inducible proteins include chitinases (Chang et al. 1995), glucanases (Krebs & Grumet 1993), proteinase inhibitors (Geoffroy et al. 1990), lipid transfer proteins (Kader 1997), proteinases (Pinedo et al. 1993), ribosome inactivating proteins (Jach et al. 1995) and several pathogenesis related (PR) proteins (Stintzi et al. 1993).

Canal systems containing various secretions, such as latices, resins, gums and mucilages, are widespread in the plant kingdom. Secretory canals appear to be ideally suited as defense against pathogens and insects (Farrell et al. 1991). Latex occurs in 12500 species belonging to 900 genera. It is a milky fluid composed of a liquid serum which holds, either in solution or suspension, a mixture of substances many of which are toxic (Lynn & Clevette-Radford 1987b). Insects and pathogens that attack latex-producing plants are normally faced with a combination of physical and chemical defenses provided by the latex. Enzymes carrying out varied functions are known to occur in various latices at high concentrations (Giordani et al. 1992, Giordani & Lafon 1993).

The various exuded latices are known to contain glycosidases (Giordani & Lafon 1993), proteases (Lynn & Clevette-Radford 1986), acid phosphatases (Lynn & Clevette-Radford 1987a), amylases (Lynn & Clevette-Radford 1987a), chitinases (Jekel et al. 1991), hevein (Van Parijs et al. 1991), proteinase inhibitors (Archer 1983, Lin & Lu 1994), b-1,3-glucanase (Chye & Cheung 1995) and various others enzymatic activities (Lynn & Clevette-Radford 1987b).

The present work hypothesizes that latex from Manihot glaziovii has antimicrobial and insecticidal properties probably due to the presence of several proteins with enzymatic and inhibitory properties.

Materials and methods

Latex of Manihot glaziovii Muell. Arg. was obtained from native and cultivated plants in the state of Ceará, Brazil. The latex was collected in a plastic recipient into distilled water (1:1 latex to water ratio), maintained at 5°C and centrifuged for one hour at 17,000 x g. The supernatant was discarded and the serum (crude latex) was recovered after freeze-drying. Solutions (10 mg.ml^-1) of this freeze-dried crude latex were employed in the assays described below except in the cases where a dialyzed and freeze dried preparation of it was used.

The activity of b -1,3-glucanase was assayed by utilizing 0.5% laminarin (Sigma Chemicals Co., St. Louis) and following reducing groups according to the method described by Fink et al. (1988). One unit of activity was defined as the amount of enzyme that produces an absorbance difference of 0.001.

Chitinase activity was measured with chitin azure (Sigma Chemicals Co., St. Louis) as substrate employing the colorimetric assay as described by Hackman & Goldberg (1964). One unit of activity was defined as the concentration of chitinase that produces an absorbance difference of 0.001.

Amylase activity was determined by the method of Mao & Kinsella (1981). The assay employed soluble starch (1%) as the substrate.

The activity of alkaline proteinases was measured by the method described by Kunitz (1947) employing casein (1%) as substrate. The activity of acid proteinases was determined on hemoglobin substrate according to the method described by Lemos et al. (1990). Cysteine proteinases activity was measured by the method of Kunitz (1947) employing azo-casein as substrate.

The measurement of lectin activity was done by a hemagglutination test utilizing rabbit blood cells as described by Pusztai et al. (1987).

The activities of both papain and trypsin were measured by comparing the activity of control solutions containing only the enzyme being assayed with that of solutions made by mixing increasing amounts of inhibitor (crude latex) with the same amount of enzyme. One unit of inhibitor activity is the amount of inhibitor that inhibits 50% of a standard preparation of enzyme as defined previously (Arnon 1970, Xavier-Filho 1974).

Inhibitory activity against a-amylase was determined by the method of Mao & Kinsella (1981) employing porcine pancreatic amylase. One unit of inhibitor was calculated as the amount of inhibitor necessary to reduce the enzyme activity by 50%.

Fungal isolates of Macrophomina phaseolina (Tassi.) Goid., Colletotrichum gloesporioides (Penz.) Penz. and Sacc., and Fusarium solani (Mart.) Sacc. (obtained from the Seção de Fitopatologia, Centro de Ciências Agrárias, Universidade Federal do Ceará, Fortaleza, Brazil) were utilized in these experiments and were maintained in Sabouraud dextrose agar at 5°C. Antifungal activity was assayed under sterile conditions using the disc-plate diffusion assay described by Roberts & Selitrenikoff (1988). Fungi were inoculated on the center of Petri dishes (8.5 cm diameter) containing Sabouraud dextrose agar. After allowing for spore germination and vegetative growth (48 hours at room temperature, 25°C) sterile filter paper discs (Whattman 3MM) were distributed on the agar surface in front of the growing fungi and 50 µl of crude and dialyzed latex solutions (6.5 mg.ml^-1 in 8 mM, pH 4.5 acetate buffer) were added to the discs. The Petri dishes were incubated for 24 hours at 25°C. A negative control was run by adding buffer instead of the latex solution. In this manner a crescent shaped zone of inhibition of fungal growth is observed around the disc if the solution being tested shows any antifungal property.

Individuals from a colony of Callosobruchus maculatus (F.) (Coleoptera: Bruchidae), the cowpea weevil (Jackai & Daoust 1986), maintained at the Departamento de Bioquímica e Biologia Molecular, Universidade Federal do Ceará, were used for feeding experiments. Artificial seeds (400 mg) made by pressing cowpea seed flour with additions (0.25%, 0.5%, 1.0%, 2.0%, 4.0%, and 8.0% of fractions from latex before and after dialysis) in a cylindrical brass mold with the help of a hand press were utilized for infestation by cowpea weevil females (Macedo et al. 1993). After infestation and eclosion, three artificial seeds with three eggs each were left for insect development in glass tubes (20 x 2 cm) covered with cotton plugs and placed in a growth chamber (25°C and 80% RH). The number of surviving insects was recorded after 40 days. The averages of two independent experiments (two replicates) were utilized to fit dose-response curves (quadratic correlation) from which we calculated the lethal dose that kills fifty percent of the animals (LD_50%) for the latex preparations.

SDS-polyacrylamide gel electrophoresis (SDS-PAGE) was done according to the method developed by Laemmli (1970). Proteins utilized as molecular marker standards for PAGE-SDS were bovine serum albumin (66 kDa), ovalbumin (45 kDa), glyceraldehyde-3-phosphate dehydrogenase (36 kDa), carbonic anhydrase (29 kDa) and a-lactoglobulin (14.2 kDa).

Protein determinations were made by the method of Bradford (1976) employing bovine serum albumin as standard or alternatively by using the absorbance at 280 nm.

Results and Discussion

There is a great interest in the elucidation of the role of latex in plants and most of the work done on those exudates is based on the assumption that they are involved in defense mechanisms of plants against insects. It is well known that latices commonly contain several enzymatic and inhibitory activities of potential action against insects and pathogens (Lynn & Clevette-Radford 1987b, Archer 1983). Assay of several enzymatic and inhibitory activity (table 1) showed that the latex of contains a-amylase, chitinase and b-1,3-glucanase activities. None of the proteolytic activities tested was present in this latex although trypsin inhibitory activity was detected. Hemagglutinating (lectin) activity was also not present. These results are in agreement with previous reports by other authors (Lynn & Clevette-Radford 1987b).

Figure 1. Effect of Manihot glaziovii freeze-dried crude latex on the survival of adult Callosobruchus maculatus. Points were fitted by computer analysis and the quadratic correlation coefficient calculated as r²= 0.981.

The effect of the M. glaziovii latex on the development of the bruchid beetle C. maculatus is shown in figure 1. We found a high negative correlation (r² = 0.981) between the amount of crude latex (6.7% protein and 19.6% carbohydrate) in the bruchid's diet and the number of adults surviving after 40 days from oviposition. An LD₅₀ of 2.9% could be calculated from the curve. In parallel experiments with the dialyzed latex (8.2% protein and 6.6% carbohydrate) we found an LD₅₀ of 1.1% and also a high correlation coefficient (r² = 0.915). These results suggest that the latex contains high molecular weight substances that strongly inhibit the development of C. maculatus.

When we employed the agar diffusion method of Roberts & Selitrenikoff (1988) to measure the antimicrobial activity of the crude and dialyzed latex we observed inhibition of growth of the phytopathogenic fungi M. phaseolina (figure 2), F. solani and C. gloesporioides (not shown) suggesting a protective role for this latex. This growth inhibition may be accounted for by the presence of the enzymes chitinase and b-1,3-glucanase and also of trypsin inhibitors which are known to be involved in defense mechanisms of plants against fungi (Shewry & Lucas 1997, Lorito et al. 1994). Crude and dialyzed latex preparations were analyzed by SDS-PAGE (data not shown) and the patterns obtained suggest that the latex of M. glaziovii is a complex mixture of proteins with molecular masses covering a range from 14 and 80 kDa. These values lie in the range of molecular weights found by others for latex proteins (Arreguin et al. 1988).

Figure 2. Agar gel plates showing growth inhibition of the fungus Macrophomina phaseolina by Manihot glaziovii latex preparations. M. phaseolina was inoculated in the center of Petri dishes (8.5 cm diameter) containing Saboraud dextrose agar and incubated for 48 h at 25C. After this incubation period filter paper discs imbibed in the test solutions (6.5 mg.ml^-1 made up in 8 mM acetate buffer, pH 4.5) were distributed on the culture medium, in front of the developing fungi. The dishes were incubated at the same conditions. The size of the halos formed around the paper discs was taken as a measure of the growth inhibition. (A) Crude and (B) dialyzed latex; (1) latex samples, (2) control buffer.

The results presented in this paper suggest that various high molecular weight compounds, possibly of protein nature, like enzymes and enzyme inhibitors and others, present in the latex of the Euphorbiaceae plant M. glaziovii strongly interfere with the development of phytopathogenic fungi and insects. The results also suggest that proteins responsible for the observed effects may potentially be utilized as insecticides or fungicides.

Acknowledgements - The work reported here was financed by the Brazilian agencies CNPq, CAPES and FINEP.

^2. Laboratório de Fisiologia e Bioquímica de Microrganismos, Centro de Biociências e Biotecnologia, Universidade Estadual do Norte Fluminense, Av. Alberto Lamego 2000, 28015-620 Campos dos Goytacazes, RJ, Brasil.

^3. Laboratório de Química e Função de Proteínas e Peptídeos, Centro de Biociências e Biotecnologia, Universidade Estadual do Norte Fluminense.

^4. Departamento de Bioquímica, Universidade Federal do Rio Grande do Norte, 59072-970 Natal, RN, Brasil.

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1. Departamento de Bioquímica e Biologia Molecular, Universidade Federal do Ceará, Caixa Postal 1065, 60001-970 Fortaleza, CE, Brasil.

. Departamento de Bioquímica e Biologia Molecular, Universidade Federal do Ceará, Caixa Postal 1065, 60001-970 Fortaleza, CE, Brasil.

Publication Dates

• Publication in this collection
  19 Aug 1999
• Date of issue
  Apr 1999

History

• Accepted
  18 Sept 1998
• Received
  13 Apr 1998