Báo cáo khoa học: "Hypoxylon mammatum toxins: possible involvement in canker development on"

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Hypoxylon mammatum toxins: possible involvement in canker development on aspen S. Rebuffat 2 I. Genetet J. Pinon B. Bodo 12 1 1 Pathologie ForestiAre, tNRACRF, Champenoux F-54280, Seichamps, and Laboratoire de 2 Appliqu6e aux Corps Organisés, CNRS UA40i, 63, Museum National d’Histoire Naturetle, Chimie rue Buffon, F-75231 Paris Cedex 05, France separated by elution of the XAD Introduction were with a methanol/water gradient: resin hymatoxins first eluted (about 40 mg/l of mammatum (Wahl.) Miller Hypoxylon filtrate) and then neutral metabolites (a stem canker on aspen (Populus causes a few mg/1). The chemical structures of tremula) and on some poplars of the Taca- these substances were determined by hamaca section (Pinon, 1976). The di- spectrometric methods (MS, IR, 1 D and sease is characterized by a flattened 2D NMR). Hymatoxins are unusual diter- sunken surface with a yellow orange mar- pene sulfates with a molecular mass of gin. H. mammatum prevents host callu- about 400. The other toxin group is consti- sing. Hubbes (1964) found that diffusible tuted of trihydroxytetralones. substances from H. mammatum agar cul- To provide a clearer understanding of tures inhibited callus formation in wounds the pathotoxic features of H. mammatum, on aspen bark. Schipper (1978) and Ster- search for and characterization of in vivo mer et aL, (1984) confirmed the possibility toxic metabolites were undertaken and the that H. mammatum could produce toxins. data are reported herein. These compounds can be isolated by par- titioning into various organic solvents and chromatography. Culture age was also supposed to affect the kind and amount of Materials and Methods metabolites produced (Stermer et al., 1984). We have isolated and character- Purification procedure ized 2 groups of toxins from culture filtrate Young aspen trees were obtained from breed- (Bodo et al., 1987). Optimum secretion ing programs (Lemoine, 1973). The trees were was achieved within 6 wk of still culture on planted and inoculated with mycelium in 1981 (Pinon et al., 1988). After 6 yr, the H. mamma- wort medium. A technique has been de- tum trees were cut. Wood and bark samples veloped to isolate the toxins from the fil- from both healthy and infected areas were col- trate by adsorption onto a neutral resin lected. Each sample was freeze-dried, ground (Amberlite XAD 4). Two groups of toxins and then kept at -20°C. 100 g of each sample
extracted in a soxhlet apparatus with 10 leaves taken for each assay. Acetone were were methanol for 6 h. The extracts were concen- used control. (1°I°) was as trated and an aliquot of each sample was tested for its toxicity using a leaf bioassay. The active were partially purified by chromatogra- extracts phy on Sephadex LH-20. The fractions obtained Results and Discussion reduced to dryness under vacuum. were When tested at a concentration of 1 Leaf bioassay mg/ml, extracts from healthy wood and bark samples had no visible effects, Throughout the purification procedure, samples bioassayed by determining their effects on were whereas those from infected wood and the leaves according to Pinon (1984). The bark samples induced necrosis. However, leaves were taken from clones obtained from the response was not as large with the in vitro cultures and grown in a greenhouse. wood extracts as compared to those of After cutting, the leaf petiole was placed in small tubes containing the fraction to test. 5 or bark.
Each toxic extract was separated into 9 demonstrated the presence of toxic sub- fractions on Sephadex LH-20. They were stances in infected bark and wood from a concentrated to dryness and 100 mg of freshly cut tree. These compounds have the concentrates were dissolved in 1 ml of no effect on the clone known for its resis- acetone, then completed to 100 ml with tance to in vitro toxins. distilled water. The fractions were bioas- Preliminary chemical examination of the sayed against 4 aspen clones which had complex toxic extract from infected bark been selected for their different behaviors does not enab!le us to conclude as to the under artificial inoculation: clones 717. presence or absence of hymatoxins and 1.2, 712.1 and 706.8 were susceptible and trihydroxytetralones. clone 710.23 was quite tolerant. The 4 clones showed the same sensi- tivity to hymatoxins and to the toxic ex- References tracts from infected trees: no necrosis was observed with clones 717.1.2 and 712.1, whereas leaf necrosis was observed with Bodo B., Davoust D., Lecommandeur D., clones 706.8 and 710.23 (Fig. 1 ). Rebuffat S., Genetet I. & Pinon J. (1987) Hyma- toxin A, a diterpene sulfate phytotoxin of Fractions 5 and 6 of the infected bark Hypoxylon mammatum, parasite of aspen. extract were always necrotic for these last Tetrahedron Lett. 28, 2355-2358 two clones, but, in addition, fraction 1 was Hubbes M. (196.4} New facts on host-parasite toxic for clone 706.8 and fraction 4 for relationships in the Hypoxylon canker of aspen. clone 710.23. From all these results, it Can. J. Bot 42,1489-1494 appears that 2 groups of toxins are pres- Lemoine M. (1973) Am6lioration des peupliers ent in the bark extract: one is eluted in the de la section Leuce sur sols hydromorphes. These, Université de Nancy I, France first fractions and the second in the middle fractions of Sephadex LH-20 chroma- Pinon J. (1976) Une menace grave pour les trembles alpins: le chancre a Hypoxylon mam- tography. When analyzed by thin-layer matum. Rev. For. Fr. 28, 31-34 chromatography, both toxic fractions were Pinon J. (1984) Proprietes biologiques de la still too complex to enable identification of toxine d’Hypoxylon mammatum, parasite des hymatoxins. peupliers de la section Leuce. Rev. CytoL Biol. V6gdt. Bot.7, 271-277 The most toxic fractions from the in- fected wood extracts were fractions 6 and Pinon J., Genetet I., Bodo B. & Rebuffat S. (1988) Apport des vitro6ulturesI’etude des 7. Each of these toxic fractions was sub- m6tabolites secondaires d’origine fongique jected to silica gel chromatography in agissant sur le cambium des peupliers. 32e order to obtain simpler fractions, allowing Colloque de la Soci6t6 frangaise de Phytopa- the search for the in vivo toxins (hyma- thologie, Angers, 14-15 mai, ACTA, in press toxins, tetralones). A.L. Jr. (1978} A Hypoxylon mamma- Schipper tum toxin responsible for canker formation in Most of the studies on toxins involved in quaking aspen. Phytopathology 68, 868-872 H. mammatum canker were carried out Stermer B.A., Scheffer R.P. & Hart J.H. (1984} with cultures of this fungus on synthetic Isolation of toxins of Hypoxylon mammatum medium. The precise role of the metabo- and demonstration of some toxic effects on lites thus isolated in pathogenesis must be selected clones