Isolation and partial characterization of phytotoxins from Curvularia lunata (Wakk.) Boed

Physi&gical Plant Path&y

(1976)

8,325-331

Isolation and partial characterization Curvularia lunata (Wakk.) Boed.?

of phytotoxins from

F. MACRO and A. VIANELLO Istituti

di

Pa&log&a Vegetalc, Univmiti

(Acceptedfw jublicatim

di.

Padova, 35100 Paabva, I&y

March 1976)

Curvuluriu lunatu (Wakk.) Boed. was found to produce at least two phytotoxins in Fries modified liquid medium which cause necrotic spots on plant leaves even when they are highly diluted. Maximum toxin production ocmrred 10 days after inoculation at 25” to 30 “C temperature and at pH 3.5. The two chromatographically isolated substances with toxic activity are soluble in water and methanol and insoluble in chloroform and diethyl ether. The-y are dialysable, heat stable and more active at acid pH. The toxic activity is lost at alkaline pH’s, but it comes back by lowering the pH to 3.5. The molecular weight of the toxin, as determined by gel filtration on Sephadex G 10, is about 350. Both the components of the toxin are fluorescent tier excitation under U.V. light suggesting the presence of aromatic rings in the molecule. The toxin is not host-specific since it damages several plant species, corn hybrids and inbred lines.

INTRODUCTION Curvuhia lunata behaves either as a saprophyte or as a pathogen on various plant species such as rice [II], corn [I, 2, 41 and sorghum [a. It may damage leaves, roots and seeds. On leaves the fungus produces small necrotic spots [I, 4, 5], in roots it causes root rot [2], while in rice seeds it invades the outer seed coat, endosperm and embryo, inhibiting germination [II]. A number of fungi which induce necrotic lesions are found to produce low molecular weight toxins [ZO, 131. C. lunuta was also reported to secrete toxic metabolites which inhibit elongation of the primary root of rice seedlings [II]. This finding suggested that C. lunata can produce phytotoxins. Aims of the present work were to: (i) veriQ the ability of the fungus to produce toxic metabolite(s) which may be phytotoxic to corn plants and other species; (ii) isolate and characterize the toxin(s) and determine some conditions favouring production. MATERIALS AND METHODS Pathogenic monoconidial cultures of C. hnata were isolated from greenhouseinoculated corn seedlings and cultured on potato-dextrose agar. Toxin was produced by still cultures at 25 “C in 250 ml Erlenmeyer flasks, each containing 50 ml of Fries medium modified to pH 3-5 and supplemented with O*l”h of yeast extract [3]. Each flask was inoculated with a small agar block from a loday-old sporulating culture of the fungus. t This

study

was supported

by the Italian

National

Research

Council.

F. Macrl and A. Vianello

326

The time-course of toxin production was studied by combining the filtrates of three flasks at 3-day intervals and assaying them for toxin production. The dry weight of the mycelial mats was also determined after they had been washed twice with distilled water and dried at 90 “C for 24 h. The influence of temperature and pH on toxin production was tested in a similar manner. Three assay procedures were used to determine toxin activity. The first was carried out according to Scheffer & Ullstrup [7]. A unit of toxin activity was defined as the reciprocal values of the assay end-point per mg of dry weight of toxic filtrate. The second was based on the development of necrotic spots caused by drops of toxin solution (20 ~1) on leaves. Each drop of toxin solution was placed on a puncture wound made with a thin needle. Injected leaves were incubated in a moist chamber at room temperature. Since the toxin seems to damage plant membranes (to be published), a third method based on inhibition of *sRb uptake by roots was used. After exposure to toxin at 25 “C for 4 h, excised roots were allowed to take up ssRb from the radioactive solution for 1 h at 25 “C. The medium consisted of O-2 mM-CaC1, solution plus O-1 mM-KC1 labelled with 0.1 @i/ml s6Rb at pH 5.6. The samples were removed and rinsed thrice for 10 min in cold 0.1 NKCl. The roots were digested at 70 “C in vials containing HsO, and radioactivity determined by scintillation counting (Packard Tri-Carb, model 3320). For all biological assays non-inoculated Fries medium at pH 3.5 was used as a control. Corn hybrids and inbred lines and other plant species were used to determine respectively relative phytotoxicity and host-specificity. The methods used for toxin purification were similar to those used by several authors [S, 91. One litre of culture liquid was filtered through several layers of cheesecloth and then through filter paper. The filtrate was concentrated to O-10 the original volume under vacuum at 45 “C. An equal volume of methanol was added, and after 48 h at 2 “C the precipitate was removed by filtration and discarded. The methanol was removed by evaporation and the remaining solution was partitioned five times with equal volumes of chloroform. The aqueous fraction was evaporated under reduced pressure at 45 “C to a red syrup. This syrup was dissolved in a small quantity of absolute ethanol, filtered, evaporated to dryness, and washed twice with small amounts of cold absolute diethyl ether. After removing diethyl ether, 100 ml of water (pH 3-5) were added. Further purification was carried out by gel filtration on Sephadex G 10 in a Pharmacia column (l-6 x 49 cm) at a flow rate of 7 ml/h. A 2-ml sample (O-07 mg/ml of toxin) was eluted with water at pH 3.5 and 5-ml fractions were collected. The toxic fraction was further purified on cellulose t.1.c. plates developed with the following solvent systems: first (a) butanol, acetic acid, water (4 : 1 : 5, upper layer) and then (b) butanol, pyridine, acetic acid, water (30 : 20 : 6 : 24). The second system was used to develop the band with R, 0.50 obtained from the first chromatogram. This band was the only one in which toxic activity was detected. RESULTS Tim-course

of toxin production

The toxin was first detected 4 days after inoculation. At this time toxic filtrate was different from Fries medium alone in root inhibition. Toxin production rose to a

Phytotoxins

of

Curvularia lunata

327

peak value 10 days after inoculation and remained approximately constant for 6 days. Mycelial dry weight followed a similar trend and remained constant, whereas the amount of toxin gradually decreased (Fig. 1).

20000 600 z E ‘ii0

16000 500

e B f’ 12000 f TJ F g s .E s I-

2 r ‘p2

400

; I”

0000 300 4000 200 1

I

I

I

I

I

4

7’

IO

13

16

IS

-r

Days FIG. 1. Time-course of toxin production (A-A) and mycelial dry weight (O----O) of C. lunatu. Toxin production measured according to Scheffer & Ullstrup [7] and mycelial mats were determined by combining three inoculated flasks of 50 ml of culture medium for each interval. The reciprocal values of the assay end point/mg dry weight of toxic filtrate were taken as activity units of the toxin per mg. The data are the mean of two replicates.

Toxin production was maximal between 25 and 30 “C after 13 days from inoculation; mycelial dry weights were also highest at the same temperatures (Table 1). Although the mycelial dry weight was lower at pH 3-5, toxic activity reached its maximum. It lost its activity when the pH of the medium rose toward higher values (Table 1). Chemical characteristics

of toxin

The toxin is soluble in water and methanol and insoluble in chloroform and diethyl ether. It is dialysable and extremely heat stable, showing no change in its activity when autoclaved for 30 min at 120 “C. The toxin is active and stable at pH 3.5, while it loses its activity at alkaline pH’s. The inactivation at high pH levels is reversible and the activity can be recovered by lowering the pH to 3.5 (Table 2). Toxic activity is retained even after 3 months when the compound is stored in the dark, in aqueous solution (pH 3.5), at 3 “C. Figure 2 shows elution pattern of the toxin obtained by column chromatography. The data of the elution were used to determine the K,” value (O-77) of the toxin and its molecular weight (approximately 25

328 TABLE

Effect of temperature

and initial pH on toxin production

Temperature (“C)

F. Macrl

and

A. Vianello

days from

inoculation

1 by C. lunata

Toxin” (units/ml)

after 13

Mycelial

dry (m&9

20 25 30 35

10 20 20 14

000 000 000 000

230 538 411 142

PH 3.5 4.0 5.0 6.0

20 000 10 000 4800 4000

680 936 1089 912

wt

Toxin production and mycelial mats were determined by combining three inoculated flasks of 50 ml of culture medium for each determination. The data are the mean of two replicates. The first method of assay quoted in Materials and Methods section was used. 0 The reciprocal values of the assay end point/mg dry weight of toxic filtrate were taken as the number of activity units of toxin per mg.

TABLE

2

Effect qf pH on toxin activity Control

Treated

PH

0,., of inhibition nmol/g

3.5 4.0 5.0 6.0 7.0 8.0 3.5a

1342 1299 1332 1341 1329 1345 1285

fresh

wt x h 799 975 1266 1345 1327 1343 765

40 25 5 40

The third assay method described in the Materials and Methods section was used. One g of roots was pretreated with toxin obtained after column chromatography (6 pg/ml) for 4 h, at 25 “C, at the specified pH’s and then allowed to absorb 0.1 m&KC1 (pH 5.6) labelled with *sRb (0.1 pCi/ml) for 1 h. Roots were rinsed three times with unlabelled 0.1 N-KC& digested and counted. 0 The toxin solution was adjusted to pH 8.0 and then lowered to 3.5 before the beginning of the experiment. The data are the mean of two replicates.

350). The column calibration was carried out with a group of substances of known molecular weight (blue dextran 2000, tri-L-tyrosine, penta-r.-alanine and potassium chromate), eluted through the same column under the same conditions used for toxin purification. Each compound eluted as a single peak, with no inflection points. The K,, values plotted against the logarithms of their molecular weights yielded a straight line (Fig. 3). The toxin (V, = 55 ml) was further purified by t.1.c. with elution in the solvent system (a). The area showing toxic activity (RF = 0.50) was isolated and rechromatographed in the solvent system (b). At least two bands with biological activity

Phytotoxins

of

Curvularia lunafa

329

Elution

volume

(ml)

FIG. 2. Fractionation of the toxin preparation and of the peptides used as standards for molecular weight determination on Sephadex G 10. A Z-ml sample (0.07 mg/ml of toxin) was eluted with water at pH 3.5 and 5-ml fractions were collected. The second assay procedure described in the Materials and Methods section was used.

. Potassium

0.8

-

C /UnUta

2 0.4

chromate

toxin

-

IO2 Molecular

FIG. 3. Molecular with 2 ml determined

weight determination of solution containing 0.5 mg/ml by spectrophotometric readings

weight

of C. lunate phytotoxin. of each compound. at 280 nm.

The The

column was loaded elution volumes were

(R, = O-20, O-55) were detected after the second chromatographic band was fluorescent after excitation at 366 nm and the second 254 nm. Biological All

characteristics

run. The first after excitation at

of the toxin

experiments were conducted by using toxin after column purification. The toxin affects corn plants even when it is at high dilution. Necrotic spots of root growth of corn seedlings were achieved with 0.3 pg/ml and 50% inhibition

F. Macrl

330

was produced with 3.5 pg/ml. The toxin damages inhibition of uptake and loss of electrolytes (to be inbred lines were tested for sensitivity to the toxin (6 in sensitivity to the toxin were detected in two of the (Table 3). TABLE

Effect

Dekalb Asgrow Marano Dekalb Funk’s

XL 342 ATC79 vicentino XL 12 G 77

A. Vianello

cell permeability determining published). Corn hybrids and pg/ml). Significant differences hybrids and inbred lines tested

3

of toxin on hybrids and inbred lines

o/0 inhibition compared to control

Hybrids

and

45a 41a 37a 36a 23b

Inbred 127.2 127.3 44.3 126.2 40.4

lines

y/o inhibition compared to control 56a 51u 48” 45” 20”

The third assay method described in Materials and Methods section was used. One g of roots was pretreated with toxin obtained after column chromatography (6 ngjml) for 4 h at 25 “C and then allowed to absorb 0.1 mu-KC1 labelled with sBRb (0.1 nCi/ml) for 1 h. Roots were rinsed three times with unlabelled 0.1 N-KCl, digested and counted. The percentage data, after conversion into angles, were subjected to analysis of variance. Values in columns not followed by the same letter for hybrids and inbred lines are significantly different at P = 0.05. The data are the mean of four replicates.

In order to determine host-specificity, tests were carried out on the following species : Hordeum vulgare L., Avena sativa L., Sorghum vulgare L., Triticum sativum Lam., Tr$olium pratense L., Medicago sativa L., Helianthus annuus L., Phaseolus vulgaris L. Necrotic spots were detected in all the above species within 4 to 5 h after treatment with 20 l,~l of purified toxin solution. DISCUSSION There is evidence that C. lunata produces in liquid culture toxins which affect corn plants. This effect is not host-specific, because many plant species and corn hybrids and inbreds are also damaged. The toxic material seems to be secreted by the actively growing mycelium of the fungus and therefore cannot be considered a product of autolysis. The molecular weight of the toxin has been determined and the fluorescent nature of the toxic compound may suggest the presence of aromatic rings in the molecule. The low molecular weight, the dialysability and the high heat stability point to a simple molecular structure of the product. Since the toxin is not host-specific, it is difficult to establish its role as a determinant of a disease [8]. According to Wheeler & Luke [12], phytotoxins are described as “products of living organism toxic to plants. The designation, phytotoxin, does not imply that such a material plays any role whatever in relation to any disease caused by pathogen”. This type of definition seems to us the most suitable way to describe the material we have extracted and purified from C. lunata cultures.

Phytotoxins

of Curvularia

REFERENCES 1. BUNTING, R. H. (1927). 2. 3. 4. 5. 6. 7. 8.

9. 10. 11. 12.

13.

luanta

331

Local cereal diseases in the records of the mycological division. Bulletin Department of Agrizulture Gold Coast Colony 7, 25-27. HYNES, H. J. (1937). Species of Helminthos#orium and Curvularia associated with root rot of wheat and other graminaceous plants. Journal of the Royal Society of .New South Wales 70, 378. LUKE, H. H. & WHEELER, H. E. (1955). Toxin production by Helminthosporium victoriae. PhytoPathology 45, 453-458. MAC&, F. & DI LENNA, P. (1974). Leaf corn blight incited by Curvularia lunata (Wakk.) Boed. Rivista di Patologia Vegetale 10, 27-35. PRAKASH, J., SAINGER, D. K., MATENR, R. S. & SUNDARAM, N. V. (1975). Resistance to five leaf-spotting fungi in forage and grain sorghums in India. Plant Disease Reporter 59, 179-183. PRINGLE, R. B. & SCHEFFER, R. P. (1967). Isolation of the host-specific toxin and related substance with non-specific toxicity from Helminthos~orium carbonurn. Phytopathology 57, 1169-l 172. SCHEPFER,R. P. & ULLSTRUP, A. J. (1965). A host-specific toxic metabolite from Helminthosporium carbonurn. Phytopathology 55, 1037-1038. SCHEPFER, R. P. & PRINGLE, R. B. (1967). Pathogen-produced determinants of disease and their effects on host plants. In The Dynamic Role of Molecular Constituents in Plant Parasite Interaction, ed. by C. J. Mirocha & I. Uritani, pp. 217-234. Bruce Publishing Co., St. Paul, Minnesota. STEINER, G. W. & BYTHER, R. S. (1971). Partial characterization and use of a host-specific toxin from Helminthoqkium sacchari on sugarcane. Phytofiathologv 61, 691-695. STROBEL, G. A. (1974). Phytotoxins produced by plant parasites. Annual Review of Plant Physiology 25,541-566. VIDHYASEKARAN, P., SUBRAMANIAN, C. L. & GOVINDASWAMY, C.V. (1970). Production oftoxins by seed-born fungi and its role in paddy seed spoilage. Indian Phytopatholog) 23, 518-525. WHEELER, H. & LUKE, H. H. (1963). Microbial toxins in plant disease. Annual Review of Microbiology 17, 223-242. WOOD, R. K. S., BALLIO, A. & GRANI~, A. (1972). Phytotoxins in Plant Diseases. Academic Press, London.