Partial Purification of Toxin from Curvularia lunata (Wakker) Boedijn

Biochem. Physiol. Pflanzen 188, 128-135 (1992) Gustav Fischer Verlag lena

Partial Purification of Toxin from Curvularia lunata (Wakker) Boedijn H.

N. GOUR,

P.

D. NITHARWAL

and

SANJEEV AGARWAL

S.K.N. College of Agriculture Rajasthan Agricultural University Jobner, India Key Term Index: Toxic metabolite, production, properties ; Curvularia lunata

Summary The fungus isolated from diseased tissues of pearl millet leaves was identified as Curvularia lunata . A toxin, isolated from 14 day old culture filtrate of C. lunata, was partially purified by ammonium sulfate fractionation followed by gel filtration through Sephadex G-IOO. The toxin was heat stable and soluble in water and acetate buffer at pH 5.5 to 6.0. Toxin was found to be most active at pH 5.5. The partially purified toxin showed some degree of host specificity. Different host varieties were screened for disease resistance . Toxin also affected cell permeability of susceptible host tissues.

Introduction

Evidence that fungal toxins have a major role in plant pathogenesis has been well elucidated. There appears to exist a balanced dialogue between production of toxins and nutritional requirement of the fungal organisms. Curvularia sp. has been reported to produce toxin which reproduces symptoms on leaves of susceptible host plants (AGARWAL and BISEN 1984). MACRI and VI ANELLO (1976) have reported a heat stable toxic metabolite from C. lunata at pH 4.0 . One of the earliest effects of some toxins is known to cause the disruption of plasma membrane of their host cell resulting in marked changes in permeability (BRONSON and SCHEFFER 1977; DAMANN et al. 1974). However, there are several mechanisms where by pathogenic microorganisms may alter host cells and induce disease. GOUR and DUBE (1985) have suggested an impairment of ion-mediating membrane ATPase enzymes by host specific fungal toxins . The present paper includes the partial purification of toxin from C.lunata and its effects on host tissues. Material and Methods Fungal/solates and Pathogenicity

The pearl millet leaves showing similar symptoms were brought to laboratory and the fungus Curvularia lunata was isolated. Identification of the fungus was confirmed by the International Mycological Institute, Kew (IMI No . 294859) . Koch's postulates were proved on BJ-I04 plants Abbreviations: CMC, Carboxy methyl cellulose; NaPP, Sodium Poly pectate; gdw, glass distilled water

128

BPP 188 (1992) 2

before the fungus was tested for its potential to secrete the toxic metabolite. For testing pathogenicity of the fungal isolate, pearl millet plants of variety BJ-104 and LCB-lO were raised from surface sterilized seeds. The inoculum consisted of spore suspension (25 - 30 conidia per low power field) which was prepared by suspending conidia in sterile water. The conidia for inoculum preparation was obtained by scrapping 7 d old culture. The spore suspension was sprayed on 2 months old plants. The inoculated plants covered in large polythene bags, pre-sprayed from inside with sterilized water, were moved to a green house where temperature fluctuated from 15 to 25°C. The initiation of spots could be observed after 72 h and plants demonstrated the characteristic symptoms after 96 to 120 h. Isolation of Toxin 100 ml Erlenmeyer flasks each containing 25 ml of Czapek's medium were inoculated with 0.1 ml of spore suspension of fungus grown in potato dextrose agar for 7 d. After 14 d of growth under stationary conditions at laboratory temperature (25 ± 2°C), the culture fluid was obtained by filtration through three layers of cheese cloth. The culture filtrate was used for bioassay. Purification of Toxin The culture filtrate was partially purified using ammonium sulfate fractionation and then gel filtration using Sephadex G-IOO column. For purification of the toxin, ammonium sulfate was dissolved, into the culture filtrate with constant stirring to get 25% saturation. The mixture was kept at 4°C for 45 min. The precipitate formed was centrifuged at 3000 x g for 20 min and discarded. To the supernatant, thus obtained, ammonium sulfate was dissolved with constant stirring to get 75% saturation. The mixture was kept at 4°C for 45 min. The precipitate thus formed was collected by centrifugation. It was dissolved in acetate buffer (0.05 M, pH 5.5). Gel filtration of the toxin was carried out using Sephadex G-100 column. The column (40 x 2 cm) was prepared, washed and eluted with acetate buffer (0.05 M, pH 5.5). The effluent was collected in 10 ml fractions at a flow rate of 20 ml h -\. Toxin activity was detected by using the detached leaf bioassay. Effectiveness of each step in the purification procedure was tested to induce typical symptoms in pearl millet leaves (BJ-104) in detached leaf bioassay. Protein Estimation Following separation on the gel column, protein content of the fractions was estimated by the method of LAYNE (1957) after reading optical density of samples in 1 cm light path cuvettes at 260 and 280 nm. For crude extracts and solutions of (NH4hS04 precipitates protein was estimated by Lowry method using BSA as standard (LOWRY et al. 1951). Sugar was estimated by Dubois method using glucose as standard (DUBOIS et al. 1951). Shoot Cutting Bioassay For bioassay of the toxin, freshly excised shoot of susceptible pearl millet (local var.) of equal weight was dipped in test tube containing toxic metabolite sample, uninoculated sterilized medium or distilled water. The symptoms produced on the leaves were recorded on 0-4 point scale: 0 - No visible spots on leaf surface, 1 - Spots covering about 25% of leaf, 2 Blighting spots covering 26 to 50% leaf area, 3 - Spots covering 51 to 75% leaf area and 4 Severe blight. Electrolyte Leakage The fresh leaf samples were used for bioassay. The leaves were cut into small pieces (l cm long and 5 mm wide) and rinsed and random samples (500 mg) were enclosed in washed cheese cloth and placed in 100 ml Erlenmeyer flasks containing 50 ml of toxin sample or distilled water on a rotary shaker for 30 min. Later the samples were removed and washed for 10 min in total with four changes of distilled water (100 ml each time). The samples were then resuspended in 50 ml of glass distilled water and replaced on the shaker. The conductance of the ambient 9

BPP 188 (1992) 2

129

solution was measured at 30 min intervals for 3.5 h with conductivity meter (Systronics, Ahmedabad). Conductance in ~m hos of the control leaves infiltrated with water was substracted from the conductance of the toxin - infiltered leaves ot determine the increase in electrolyte leakage induced by toxin (KOHMOTO et al. 1979). All the experiments were repeated three times.

Results Partial Purification of Toxin

Toxin responsible for leaf blight disease of pearl millet was partially purified from C. lunata using ammonium sulfate fractionation and gel filtration using Sephadex G-IOO

column chromatography (Fig. 1). The effectiveness of each step in removing contaminat-

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Fig. 1. Purification of Curvularia lunata toxins using Sephadex G-lOO. Culture filtrate was purified using (NH4hS04 fractionation. Ammonium sulfate fractions were passed through .), protein profile Sephadex G-lOO column and fractions were assessed for toxin activity (. (•....•) and sugar profile (6----6).

ing materials is presented in Table 1. The biological activity of the toxin increased with each step of purification. Specificity of Toxin

The partially purified toxin from the isolated strain induced characteristic spots which were similar to those collected from naturally infected plants. Different varieties were screened against the fungus C. lunata and its partially purified toxin (Table 2) and on the basis of symptom rating produced by toxin were termed as tolerant, semi-tolerant, 130

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Table 1. Summary table of purification of toxin from Curvularia lunata culture filtrates. Step

Procedure

Volume (ml)

Toxin activity rating*

Total protein (Ilg)

1. 2. 3.

Culture filtrate (NH4hS04 fractionation Gel filtration on Sephadex G-100 1. Peak I II. Peak II

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Total sugar (Ilg) 30.000 1.960 422 142

* The symptom rating was done after 24 h on 0-4 point scale where, 0 = no symptom, 1 = very poor symptoms, 2 = poor symptoms, 3 = good symptoms, 4 = very good symptoms.

Table 2. Screening of pearl millet varieties against the partially purified toxin and Curvularia lunata. S. No.

1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14.

Varieties

LCB-lO BJ 104 CJ 104 BGP 725 BGP 727 Baj. (Local) BGP 728 BGP 729 BGP 730 BGP 731 BGP 732 UU 5 (J) MP 124 BK 560

Symptoms rating* With purified toxin after 48 h

With C. lunata after 120 h of inoculation

4 HS 4 HS 2 ST 3S 2 ST 4 HS 3S 1 ST 4 HS 3S 1 ST 3S OT 3S

4 HS 4 HS 1 ST 3S 1 ST 4 HS 3S 1 ST 4 HS 3S OT 3S 1 ST 3S

T=Tolerant (0); ST=Semi tolerant (1-2); S=Susceptible (3); HS=Highly susceptible (4) * Symptom rating was done on 0-4 point scale, where, 0 = No symptom, 1 = Very poor symptom, 2 = poor symptom, 3 = Good symptom, 4 = Very good symptom

susceptible and highly susceptible. Variety MP 124 was resistant, BGP 732, CJ 104, BGP 727 were semi-tolerant and LCB-10, BJ 104, pearl millet local, BGP 730 were most susceptible varieties. Host range of the fungus and activity of the toxin in different crop were assessed by shoot cutting bioassay method. Shoot cuttings of six different locally available crop plants were tested for host specificity. C.lunata could infect pearl-millet, botde-gourd, cowpea and kidney-bean but could not infect other hosts like cluster-bean, chilli and field-bean (Table 3). 9*

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Table 3. The host specificity test of partially purified Curvularia lunata toxin on different field crop plants. S. No.

Field crop plants

Symptom rating*

1.

Cluster bean (Cyamopsis tetragonolobus (L.) Taub.) Bottle gourd (Lagenaria siceraria Schwein.) Cowpea (Vigna unguiculata (L.) Walp.) Kidney bean (Phaseolus aconitifolius Jacq.) Chilli (Capsicum annuum. L.) Field bean (Dolichos lablab L.) Pearl millet (Pennisetum americanum (L.) K. Schum.) (Host plant)

0 3 2 2 0 0 4

2. 3. 4. 5. 6. 7.

* The symptom rating was done on 0-4 scale where, O=No symptom, I = Very poor symptom, 2 = Poor symtpm, 3 = Good symptom, 4 = Very good symptom. 2·0

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Properties of Toxin

The toxin was partially soluble in water but readily soluble in acetate buffer at pH 5.5. to 6.0 and insoluble in organic solvents like toluene, acetone, ethyl acetate and ethyl alcohol. The biological activity of toxin was found to be max~ imum when dissolved in acetate buffer at pH 5.5. to 6.0. Toxin is heat stable 132

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Fig . 3. Permeability alterations of Pearl millet leaf using Curvularia lunata toxin. Fractions collected from Sephadex G-IOO column were tested on host leaf for ion-leakage using conductivity meter. Symbols: ~ Fraction No . 5, 0--0 Fraction No . 6,. • Fraction No . 7, fs----.:L Fraction No. 8, Fraction No . 9, . - - . Fraction No. 10, 0--0 Control.

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and its biological activity was not affected by boiling for 20 min. The UV absorption spectrum of partially purified toxin is presented in Fig. 2. The maximum absorbance was at 220 nm. Permeability Alteration

The effect of toxin on permeability of host plant was tested using a conductivity meter. Toxin fractions (Fraction No. 6 to 10) showed a distinct effect on loss of ions from leaves treated with partially purified fractions. The leakage pattern from different fractions treated samples resembled to that symptom developed on the leaves. Fraction No. 6 caused a greater ionic leakage followed by fraction No. 9 (Fig . 3). Evidently both these fractions contained high toxicity. Other fractions viz. 5, 7, 8 and 10 behaved more or less identically also showing losses of electrolytes. BPP 188 (1992) 2

133

Discussion

Curvularia lunata (Walker) Boedijn, manifested the disease symptoms on leaves of pearl millet (local) plants, identical to those collected from field conditions in pathogenicity tests, suggests that the fungus isolated was virulent and pathogenic. Toxin was partially purified by ammonium sulfate fractionation followed by gel filtration through Sephadex G-IOO. The partially purified toxin expressed the disease symptoms. The two peaks of toxin suggest that there may be two types of toxins produced by C.Zunata. Toxin is found to be heat stable. MACRI and VIANELLO (1976) also observed the similar findings with C. lunata. The toxin from V. dahliae was found to be heat stable (GOUR and DUBE 1985). The purified toxin did not exhibit host specificity and therefore it is termed as nonspecific phytotoxin, as the toxin is capable of producing some disease symptoms on susceptible host and some non-host plants. Changes in cell permeability is one of the striking events in pathogenesis. The cause of permeability alterations in most of the plants disease has been attributed to the toxin metabolites. There is considerable evidence that some susceptible tissues exhibit greater permeability than resistant when infected by parasites (WHEELER 1976). The two purified fractions of toxin (6 and 9) greatly altered the cellular permeability of susceptible pearl millet tissue. These findings were perfectly in concordance with the earlier work done with host selective and non-host specific fungal toxins. Toxin can provide an efficient means to screen available varieties for resistance. The purified toxin fractions were used in screening some available varieties and were compared with fungus produced symptoms . Interestingly, largely the symptoms produced by fungus and toxin were comparable . Theses studies suggest the capability of fungus C.lunata to secrete toxic metabolites, which induce disease symptoms on susceptible pearl millet plants and also cause alterations in cellular permeability. Acknowledgements Authors thank IMI , Kew for identifying the culture and to the authorities of SKN College of Agriculture, Jobner for providing laboratory facilities.

References AGARWAL, G. P., and BISEN , P. S.: Toxin production by Curvularia lunata var. aeria causing leaf blight in French bean (Phaseolus vulgaris). J. Ind. Bot. Soc. 63, 292-297 (1984). BRONSON , C. R., and SCHEFFER , R. P.: Heat and aging induced tolerance of sorghum and oat tissues to host selective toxins . Phytopathol. 67,1232-1238 (1977) . DAMANN , K. E. , jr.; GARDNER, J. M ., and SCHEFFER, R. P.: An assay for Helminthosporium victoriae toxin based on induced leakage of electrolytes from oat tissue. Phytopathol. 64, 652-654 (1974). DUBOIS , M., GILLES , K., HAMILTON, J. K., REBERS , P. A., and SMITH , F.: A colorimetric method for the determination of sugare . Nature 168, 167 (1951) . GOUR, H. N., and DUBE , H. c.: Effects of Ouabain and phytotoxic metabolites from Verticillium dahliae on the cell membranes of cotton plants. Physiol. Plant Pathol. 27, 108-118 (1985) . KOHMOTO, K., SCHEFFER, R. P. , and WHITESIDE, J. 0.: Host selective toxins from Alternaria citri. Phytopathol. 69, 667-671 (1979). 134

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LAYNE, E.: Spectrophotometric and turbidometric methods for measuring proteins. In: Methods of Enzymology, Vol. III (Ed. COLOWICK and KAPLAN) Academic Press Inc., New York and London 1957. LOWRY, O. H., ROSEBROUGH, N. J., FARR, A. L., and RANDALL, R. J.: Protein measurement with the folin phenol reagent. J. BioI. Chern. 193, 265-275 (1951). MACRI, F., and VIANELLO, A.: Isolation and partial characterization of phytotoxins from Curvularia lunata (Wakk.) Boed. Physil. Plant Pathol. 8, 325-331 (1976). WHEELER, H.: The role of toxins in specificity. In: Specificity in Plant Disease (Eds. WOOD, R. K. S., and GRANITE, A.) pp. 217-230, Plenum Press, New York 1976. Received June 14, 1991; revised form accepted October 14, 1991 Authors' address: Dr. H. N. GOUR, P. D. NITHARWAL and Dr. S. AGARWAL, Departments of Plant Pathology and Biochemistry, SKN College of Agriculture, Rajasthan Agricultural University, Jobner, Rajasthan 303329, India.

Buchbesprechung GISI, U., SCHENKER, R., SCHULIN, R., STADELMANN, F. X., und STICHER, H.: Bodenokologie. Reihe: Flexible Taschenbiicher, XI, 304 S., 142 Abb. u. 51 Tab. Georg Thieme Verlag-, StuttgartNew York 1990. Preis: brosch. DM 36. Unter Hinweis am SchluB des vorliegenden Taschenbuches, daB der gegenwiirtig in der Offentlichkeit viel diskutierte Bodenschutz letzten Endes "eine Sache des UmweltbewuBtseins der Bevolkerung" ist, wird unter dem Titel "Bodenokologie" ein ausgezeichneter, zusammenfassender Uberblick iiber die Teildisziplinen der Bodenkunde und die dem Begriff Okologie entsprechenden Interaktionen gegeben. Einer kurzen EinfUhrung in die Begriffsbestimmung der Bodenokologie schlieBen sich 5 iibersichtlich gestaltete und mit einpriigsamen, demonstrativen Abbildungen versehene Hauptkapitel an. In ihnen werden von groBer Sachkenntnis gepriigte iiberaus priignante AusfUhrungen gemacht: zur Zustandsbeschreibung des Bodens mit einem umfangreichen Unterkapitel zum Edaphon, zu den im ungestOrten Boden ablaufenden physikalischen, chemischen und biologischen Prozessen mit iibersichtlicher Darstellung der Stoffkreisliiufe von C, N, P und S und zur Bodenfruchtbarkeit, zu den positiven und negativen Auswirkungen menschlicher Eingriffe in den Boden (Bodenbearbeitung und Bodenbewirtschaftung, Diingung, Pflanzenschutzmittel, Schadstoffe) und abschlieBend zu Problemen des unbedingt notwendigen Bodenschutzes in verschiedener Hinsicht. 1m Unterkapitel Diingung hiitten vielleicht einige AusfUhrungen zur Errnittlung der Niihrstoff- und Diingerbediirftigkeit von BOden und Pflanzen die Darlegungen bereichem konnen. Die verwendete Literatur ist yorteilhaft sachgebietsweise zusammengefaBt. Bin urnfangreiches Sachverzeichnis erleichtert die Benutzung des Taschenbuches als ein in jeder Weise zu empfehlendes Informations- und Nachschlagewerk fUr Studenten an Hoch- und Fachschulen, Dozenten, Wissenschaftler, Berater und Fachkriifte der Land- und Forstwirtschaft, des Gartenbaues, der Landespflege, des Umweltschutzes und fUr alle 6koiogiebewuBten Interessenten. W. Bergmann, Jena BPP 188 (1992) 2

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