Physiological Plant Pathology (1982) 20, 313-319 Identification of 3(methylthio) propionic acid as a blight- inducing toxin produced by Xanthomonas campestris pv. manihotis in vitro I). PERREAUX, H. MARAITE and J. A. MEYER Laboratoire de Phytopathologie, Universite’ Catholique de Louvain, 3 Place Croix du Sud, S: 15 DO, B-1348 Louvain-la-Neuve, Belgium (.lcceptedfor publication January 1982) A toxic substance was extracted by ethyl acetate from a broth culture of Xanthomonas campestris pv. manihotis acidified at pH 4 with 1 N HCl. When infiltrated into cassava leaves the crude extract produced blight symptoms similar to those induced by the bacteria during patho- The toxic substance was purified by gel filtration on a Sephadex GlO column (Pharmacia) and by thin layer chromatography on silica gel 60 (Merck), where it showed a RF of 0.6 using as migration system methanol : chloroform : n-hexane (3 : 2 : 1, v/v). By mass spectrometry, infrared spectroscopy, and C, H, N analyses the purified toxin was identified as S(methylthio) propionic acid. This identification was confirmed by chemical synthesis. On detached cassava leaves, 3(methylthio) propionic acid gave a toxic effect up to a concentration of 9.9 mM corresponding to 24 pg of pure substance in a 20 1.11 drop of water. INTRODUCTION Bacterial blight of cassava (Manihot esculenta Crantz), caused by Xanthomonas campestris pv. munihotis (Berthet & Bondar 1915 [I]) Dye 1978 [3] (I.S.P.P. 1980 [4-j) is one of the most devastating and widely distributed diseases of cassava [Z]. It is characterized by water-soaked angular leaf spots often enlarging under high humidity into large discoloured areas becoming necrotic in a few days. The rapidity of extension of these areas and the fact that the bacteria generally cannot be isolated from them, suggest the involvement of toxic substances diffusing from the angular spots into the laminar tissues. Colonization of the stem tissues leads to the wilting of the leaves and tip die-back. Histological studies have shown bacteria in the xylem vessels [ 181, collapse of the vessels [7j and the formation of lytic pockets in the xylem, phloem and cortex parenchyma. However, in plants showing the first wilt symptoms, an important proportion of vessels appeared to be unaltered [14]. Toxic substances could thus also be involved in wilt initiation. Blight symptoms, similar to those induced by the bacteria, have been reproduced with extracts from a broth culture of X. campestris pv. manihotis [I.?]. This paper reports the purification and identification of the toxic compound. MATERIALS AND METHODS ,?reparation of a crude toxic extract The Brazilian isolate BM 12 (ICPB XM 147), provided by Dr A. L. G. Pereira, tnstituto Biologico, Sao Paulo, particularly virulent in greenhouse inoculations [S], 004%4059/82/0303 13 +07 $03.00/0 @ 1982 Academic Press Inc. (London) Limited