Glutinosin: a fungistatic metabolic product of the mould Metarrhizium glutinosum S. Pope

Abstract

Culture media on which the fungus Metarrhizium glutinosum has grown are highly toxic to other fungi. The fungus grows well on such standard synthetic media as Czapek-Dox or Raulin-Thom, but use of a crude grade of glucose in stead of pured extrose encourages sporulation and slightly increases the fungistatic activity of culture filtrates. It has been shown that yeast extract, biotin and aneurin supplements to a pure dextrose medium produce a similar response. In a study of the nature of the nitrogen source in the culture medium in relation to production of fungistatic culture filtrates, it has been found that nitrate nitrogen is very effective for growth of the fungus but not very effective for production of the fungistatic substance. Inorganic ammonium salts are ineffective in both respects, but peptone or ammonium tartrate were found to be highly effective both for growth and production of the fungistatic substance. This superiority of ammonium tartrate led to an investigation of the relation between organic acids and ammonia assimilation. Addition of a variety of organic acids, inconcentrations of 0.05 to 1.0%, to an ammonium sulphate medium, led to great increases in growth and in production of the fungistatic substance. These acids included tartaric, malic, succinic, malonic, oxalic, citric, acetic, glycollic, pyruvic, aspartic and glutamic acids. These acids do not stimulate when added to a nitrate, amino-acid or peptone medium. Various possible explanations of this effect have been considered, and it is suggested that M. glutinosum is unable, when nitrogen is supplied as an ammonium salt, to break down glucose to the 3-, 4- or 5-carbon acids which normally enter into the transformations of the Krebs cycle. Accordingly, this fungus is unable to assimilate ammonia, which is probably normally, in fungi as in other plants, condensed with α -keto acids, produced by carbohydrate degradation, to form corresponding α -amino-acids. When these acids, or acids which could probably be transformed into such acids in the cell, are supplied in the nutrient medium, growth is accordingly increased. The active fungistatic material, glutinosin, can be extracted from culture filtrates by extraction with ether, petrol-ether or benzene, the latter being most effective; more effective still is treatment of the culture filtrate with charcoal followed by elution of the charcoal with hot benzene. One vaporation of the solvent and recrystallization of the residue from ethyl alcohol, glutinosin is obtained in pure crystalline form; analyses and molecular weight determinations indicate that glutinosin has the molecular formula C 48 H 60 O 16 . Glutinosin is highly toxic to many fungi but is markedly specific; germination of spores of Botrytis allii in Czapek-Dox is inhibited by 0.8/ μ g./ml., but Trichoderma viride is not in ­ hibited by 50 μ g./ml. It is at most only slightly toxic to bacteria. Aqueous solutions are relatively stable, even when heated, but are less stable when alkaline than when acid. A volatile metabolic product of Metarrhizium glutinosum causes a severe dermatitis; the dermatitic substance is extracted by the same organic solvents as used for the extraction of glutinosin, but it is not identical with glutinosin.