Structures, properties and relationship to the aspergillomarasmines of toxins produced by Pyrenophora teres

Physiological P&n: Pathology (1979) 14,41-46

Structures, properties aspergillomarasmines teres

and relationship to the of toxins produced by Pyrenophora

E. BACH, S. CHRISTENSEN, L. DALGAARD, P. 0. LARSEN, C. E. OLSEN Chemistry Department, The Royal Veterinary and Agricultural University, Thorvaldrensvej 40, DK-1871 Cojenhagen V, Denmark

and V. SMEDEGARD-PETERSEN Department of Plant Pathology, The Royal Vet&nary and Agricultural University, Thorvalafsensvej40, DK-1871 CopenhagenV, Denmark (Acceptedfor publication

3unc 1978)

The structures have been determined of toxins A, B and C, all isolated from a cell-free sterile culture filtrate of Pyrcnophora &es. Toxin A was N-(2-amino-2-carboxyethyl)aspartic acid, not previously known from natural sources. Toxin C was identical with aspergillomarasmine A, and toxin B, probably an artefact, was identical with anhydroaspergillomarasmine A. Toxin A and Cwere toxic at a concentration of 0.25 mmol l-r, whereas toxin B was toxic only at a concentration of 1 mm01 1-l. The results support a wide distribution of toxins of the aspergillomarasmine group, but since systematically closely related plants reacted differently towards the pure toxins, a high specificity must bc involved in their action.

INTRODUCTION Pyrenophora teres Dresch. invades barley (Hordeum vulgare L.) and causes the net-spot blotch disease. The fungus, both when grown in liquid medium and in intact barley plants, has been shown to produce two toxic compounds, toxin A and B, which may be partly or wholly responsible for the pathological changes taking place in the plants [9, 101. We now report the results of repeated isolations and of chemical studies to establish the structure of the toxins produced by P. teres.

MATERIALS AND METHODS The isolates of P. teres and the production of toxins in culture have been described previously [9, 101. The bioassay employed here was a variation of that previously performed using excised leaves from 12- to 14-day-old barley seedlings. To avoid pH effects of the acidic compounds involved, these were dissolved in O-022 M phosphate buffer, pH 6.5. Blank tests were run using the same buffer. To avoid salt damage the buffer was very weak and thus the pH was not the same in the samples with varying toxin concentrations. It is, however, reasonable to assume that the difference in pH did not influence the results obtained, especially in the most dilute, biologically active solutions where the pH was nearly the same. 0048~59/79/0101-0041$02.00/0

@ 1979 Academic Press Inc. (London) Limited

42

E. Bach

et al.

The isolation procedure used was a variation of that described previously (9, lo]. Sephadex columns and acidic ion-exchange resins were not used, the main element in the procedure being chromatography of acidic amino acids on a strongly basic ion-exchange resin in the acetate form. Elution was carried out with aqueous acetic acid and aqueous formic acid at a temperature below 5%. The compounds were obtained after lyophilization as white, crystalline or amorphous solids. The purity of the isolates was established by paper chromatography (PC) and high voltage paper electrophoresis (HVE) as well as by lH- and isC-nuclear magnetic resonance spectroscopy (‘H- and 13C-NMR), mass spectroscopy (MS) an d combined gas chromatographymass spectroscopy (GGMS). The spectroscopic methods proved that no organic impurities were present; high-resolution MS established the elemental compositions. ‘H-NMR was performed at 270 MHz on a Bruker HX 270 and at 90 MHz on a Bruker HX 90E instrument. 13C-NMR was performed at 22.63 MHz on a Bruker WH 90 instrument. MS and GC-MS were performed on an AEI 3074 spectrometer. The compounds were transformed to methyl esters, to N-acetylated methyl esters and to trimethylsilyl derivatives before MS. HVE was performed at pH 3.6 (pyridine : acetic acid : H,O, 1 : 20 : 200) (buffer 1) and at pH 6.5 (pyridine : acetic acid : H,O, 25 : 1 : 500) (buffer 2) on Whatman No. 3 MM paper using a flat-plate unit (Shandon 1.24). Optical rotations were measured on a Perkin-Elmer Model 141 photoelectric polarimeter in 1 dm tubes. PC and treatment with strong acid were performed as described previously [9, 101. Samples of aspergillomarasmine A and B were available for comparison through the courtesy of Dr M. Barbier, Institute de Chimie des Substances Naturelles, CNRS, Gif-sur-Yvette, France.

RESULTS

Previously toxins A and B have been isolated from P. teres[9, 101. The new isolations provided toxin A and a third toxin, provisionally called toxin C. Toxin B was obtained only in trace amounts and was probably produced previously by nonenzymatic ring closure of toxin C. The combined chemical evidence has permitted the establishment of the formulae shown in Fig. 1. Toxin A is thus N-(2-amino-2-carboxyethyl)aspartic acid, a compound previously undescribed from natural sources. Toxin B is I-(e-aminoP-carboxyethyl)-6-carboxy-3-carboxymethyl-2-piperazinone, previously partially characterized by Haenni et al. [S] and named anhydroaspergillomarasmine A. Toxin C is N-[2-(2-amino-2-carboxyethylamino)-2-carboxyethyl]aspartic acid, previously described as aspergillomarasmine A by Haenni et al. [6]. The chemical evidence comprises high-resolution MS giving the correct elemental composition of the three compounds and degradation patterns in agreement with the structures and with the spectra of the ethyl esters of aspergillomarasmine A and anhydroaspergillomarasmine A previously reported by Haenni et al. [S]. The lHand 13C-NMR spectra are in complete agreement with the structures exhibiting signals for all C-atoms and for all H-atoms, except those exchangeable with water. The lH-NMR spectra were recorded at different ionization stages of the compounds, permitting also the determination of titration shifts and all coupling constants.

43

Toxins of Pyrenophora teres coj I +

coj CH&H+H

H’F.‘NH3 CH&H&H COj

H3b

1

CH2

c *H

c ol

Llj

iO2H

3

COZH 2

+ ,H,-N,,~;: H02C-H+-iH24-H I coj

HN & 2 -

-H2C-NH2-

CH2-kH? +

I CO2H

4

c 4-l

!Oj

‘i(3 -H

F F”’ C02H

!i

FIG. 1. Structures of P ’enq’huru terestoxins and related compounds:

1, toxin A; 2, toxin B (anhydroaspergillomarasmineii) ; 3, toxin C (aspergillomarasmine A) ; 4, aspergillomarasmine B (lycomarasmic acid) ; 5, lycomarasmine.

Acid degradation of toxin A gave, as expected, aspartic acid as the only ninhydrinreacting compound. Acid degradation of toxins B and C gave aspartic acid, diaminopropionic acid and minute amounts of alanine in agreement with the results reported for aspergillomarasmine A and anhydroaspergillomarasmine A [S] . Toxin B may be produced from toxin C by treatment with acid at room temperature, as demonstrated by lH-NMR spectroscopy (the reaction is completed after a couple of days in 1 N trifluoracetic acid). The identity of toxin C as aspergillomarasmine A was finally established by comparison with the authentic sample. The behaviour in PC and HVE was identical, and the lH-NMR spectra were indistinguishable. For toxin C [x] g-45” (c = 0.5, phosphate buffer pH 7,0*067 M) was found in good agreement with the value reported for aspergillomarasmine A ([a] g - 48”, c = 1, phosphate buffer, pH 7) [6J. Since Haenni et al. [S] have established that the configuration of the asymmetric centre in the aspartic acid moiety is L whereas the two centres in the diaminopropionic acid moieties have n-configurations, the same must apply for toxin C as depicted in Fig. 1. Since toxin B is derived from toxin C, the configuration of the asymmetric centres in this compound must be the same. For toxin B it was found that [ct]E -26” (c = 0.8, phosphate buffer pH 7, 0.067 M). For toxin A it is assumed, by analogy, that there is L-configuration in the aspartic acid moiety, and n-configuration in the diaminopropionic acid moiety, as depicted in Fig. 1. For toxin A it was found that [a]: - 19” (c = 1.0, phosphate buffer pH 7, 0.067 M). The structure proposed for toxin A is in agreement with all the spectroscopic and degradative evidence. Furthermore, preliminary experiments have shown that toxin A can be synthesized by addition of racemic diaminopropionic acid to maleic acid. This gives a mixture of two racemic diastereoisomers, which can be separated by

E. Bach et al.

44

crystallization from water. The most soluble isomer has the same lH-NMR spectrum, Rp value in PC and mobility in HVE as toxin A, and the MS of the trimethylsilyl derivatives are similar. The syntheses of the mixture of diastereoisomers by more complicated routes have previously been reported [3, 71. The R, values for toxin A, B and C in PC and the mobility in HVE relative to aspartic acid are reported in Table 1. Further details of the chemical results obtained will be presented elsewhere. TABLE 1 R, values in pajer chromatography (PC) and mobilify in high v&g6 pa@ electrophoresis(HVE)

for toxinsfrom Pyrenophorateres PC. Whatman

No. 1

HVE, Whatman No. 3 MM: mobility relative to aspartic acid Buffer 1 Buffer 2 (PH 3.6) (PH 6.5)

paper, solvent butanol : acetic acid : water (12 : 3 : 5) RF 0.09 0.06 0.05

Toxin A Toxin B Toxin C

I.16 l-46 1.84

o-93 1.09 1.18

TABLE 2 Toxic+ of toxins from Pyrenophora teres tu lcavcs of bmky (Hordeum vulgare)

Toxin A Toxin B Toxin C Synthetic toxin Aa

4

2

1

0.5

1 3 4

4 1 2 3

4 1 2 3

2 1 l-2 3

Concentration 0.25 1 o-1 O-l 1

(mu) 0,125

0.063

o-031

-

-

-

1

1

1

Rating scale: 0, no symptoms; 1, slight chlorosis; 2, marked chlorosis, slight necrosis; 3, marked chlorosis and necrosis; 4, extensive chlorosis and necrosis, eventually watersoaking and collapse of tissue. -, Not determined. 0 Synthetic mixture of diastereoisomeric racemates.

The results of the bioassay are presented in Table 2. For comparison, the synthetic mixture of diastereoisomers of toxin A has been included. It is obvious that toxin B is less effective than toxins A and C. There is, however, no doubt that a toxic effect is also present in toxin B. DISCUSSION

Both aspergillomarasmine A and the closely related compounds aspergillomarasmine B (lycomarasmic acid) and lycomarasmine (Fig. 1) have previously been isolated from various fungi pathogenic towards higher plants. Lycomarasmine has been isolated from Fusarkm lycopersici Sacc. which attacks tomatoes (Lycofiersicum esculentum) [S]. Aspergillomarasmines A and B have been isolated from Aspargillus frauus otyze [2, 6-j and aspergillomarasmine A from Fusarium oxysporumf. sp. melonis

Toxins of Pyrenophora teres

45

[11]. Aspergillomarasmine A has been isolated from the strain of Colletotrichum gloeosporioidesPenz. which is pathogenic on willows (Sulix) [4], whereas aspergillomarasmine B has been isolated from the strain of C. gloeosporioideswhich is pathogenic on olive trees (Olea europaeL.) [I, 21. N ow aspergillomarasmine A has been isolated from a new fungus, and is probably involved in the infection of barley by that fungus. The group of toxins is, therefore, widely distributed and involved in many fungus-plant interactions. The toxicity of aspergillomarasmines A and B and lycomarasmine has previously been studied using the wilt test of tomato leaves [Z]. All three compounds, and also a synthetic mixture of the two diastereoisomeric racemates of toxin A, are active in this test [2, 31. Lycomarasmine is also toxic to Solarium tuberosum,Pelargonium zonale and Vitis vinifera. Lycomarasmine as such is stated not to be toxic to Gossypium herbaceum, Impatiens holstii, Lupinw polyphyllus, Phaseolus vulgaris, Pisum sativum or Ricinus communis, but the iron-complex of lycomarasmine is toxic to these species. Neither free lycomarasmine nor the iron-complex is toxic to Sparmania africana [5j. Aspergillomarasmine B is toxic to olive trees [I, 21 and aspergillomarasmine A to willows [4]. A mixture of toxin A and B from Pyrenophorateresis toxic not only to barley but also to rye (Secalecereale),wheat (Triticum aestivum) and tomato. A number of other species, including oats (Avena sutiva), various grasses, Medicago sativa and TrijXum pratense,were not affected by toxins A+B [9, 101. The range of plants affected is thus large, but systematically closely related species react differently. This latter fact cannot be ascribed to differences in the plant-fungus interrelationship, since the experiments were performed with pure compounds. According to Gaumann [5J, the toxic action is related to the complexforming ability of the toxins. The complex-forming ability is easily observed by an inspection of the formulae of the compounds (Fig. l), which all contain at least one ethylenediamine group to which is attached carboxyl groups. In toxin B one of the amino groups in the ethylenediamine structure is acylated and thus without basic properties and with highly reduced complex-forming abilities. It is, therefore, to be expected that this compound has only low toxicity. It seems likely that toxin B is an artefact, mainly produced during the isolation of the toxins. However, it must to a certain degree be produced from toxin C in the weakly acidic culture medium by non-enzymatic transformation. This corresponds to the accumulation of anhydroaspergillomarasmine B in the culture medium of Aspergillus JIavus oryzae producing aspergillomarasmine B and in the culture medium of Fusarium lycopersici Sacc. producing lycomarasmine [2, 61. Toxin A from Pyrenophoru&es has a simpler structure than the other toxins in the group. This will probably permit synthesis, thus making the compound available in substantial amounts for use in experiments on specificity and resistance. As mentioned above, preliminary experiments have indicated the possibility of such a synthesis from maleic acid and diaminopropionic acid. This synthesis probably will also permit the isolation of the four stereoisomers, thus permitting investigations on the stereospecificity of the action of the toxin. Furthermore, an adaptation of the synthesis to produce 14C- and sH-labelled material will open up new possibilities for studies of the biochemical reactions involved in the toxic action.

46

LBaehdal.

The investigations were supported by grants from the Danish Agricultural and Veterinary Research Council and the Danish Natural Science Research Council. REFERENCES 1. BALLIO, A., E~OITALICO, A., BUONOCORE,V., CAIULLI, A., Dr Vrrroaro, V. & GRANITI, A. (1969). Production and isolation of aspergillomarasmine B (lycomarasmic acid) from cultures of Colle&otrkhumgloeosporiaidesPenz. (Glocosporium olivarium Aim.). Phytopathologia Meditewanea 8, 187-196. 2. BARBIER, M. (1972). The chemistry of some amino-acid derived phytotoxins. In Phytvtaxins in Plant Diseases, Ed. by R. K. S. Wood, A. Ballio and A. Graniti, pp. 91-103. Academic Press, London, New York. 3. B~CDANOVSKY,D. & BARBER, M. (1964). Syntheses d’analogues de la lycomarasmine et des aspergillomarasmines : acide amino-2-aspartyl-3-propionique et acide diaspartyl-2,3propionique. Bulletin a? la Soci& Chimique dc France 2778-2782. 4. BOUSQUET,J., VEGH, I., POUTEAU-THOUVENOT, M. & BARBIER, M. (1971). Isolement de l’aspergillomarasmine A de cultures de Colletotrkhum gloeos@ioiaks Pens., agent pathogtne des saules. Annales de Phytopathologic3, 407-408. 5. G&MANN, E. ( 195 1). Some problems of physiological wilting in plants. Advancesin En~logy 11, 401-437. 6. HAIXNNI, A. L., ROBERT, M., VEER, W., Roux, L., BARBIER, M. & LEDERER, E. (1965). Structure chimique des aspergillomarasmines A et B. Helvetica Chimica Acta 48, 729-750. 7. HARDEGGER,E., ANDREATTA, R., SZABO, F., ZANKOWSKA-JASINSXA, W., ROSTELLER,C. & KINDLER, H. (1967). Welkstoffe und Antibiotika. 36. Mitteilung. Synthese von Verbmdungen der Lycomarasmin-Reihe. Helvetica Chimica AC& 50, 1539-1545. 8. ~RDEGGER, E., LIECHTI, P., JACKMAN, L. M., BILLER, A. & PLANNER, P. A. (1963). Welkstoffe und Antibiotika. 24. Mitteilung. Die Konstitution des Lycomarasmins. Helvetica Chimica Acta

46,60-74. 9. SYEDEG~D-PETERSEN,V. (1976). Pathogenesis and genetics of net-spot blotch and leaf stripe of barley caused by Pyrenophora teres and Pyrcfiophoragraminca. Dissertation, DSR Forlag, Royal Veterinary and Agricultural University, Copenhagen, 1-176. 10. SMEDEG~RD-PETERSEN, V. (1977). Isolation of two toxins produced by Pyrenophora ties and their significance in disease development of net-spot blotch of barley. Physiological Plant Pathology 10, 203-211. 11. TROUVELOT, A., CAMPOROTA,P., BARBIER, M. & POUTEAU-THOUVENOT,M. (1971). Isolement de l’aspergillomarasmine A du milieu de culture de Fusarium oxysporum f. sp. mclonis. ComprCs RendusHebdomadaires&s Shancesde I’Aca&mie &s Sciences,&it D: Sciences.Naturelles, 272, 754756.