The production of viridin by pigment‐forming strains of Trichoderma viride

Abstract

Viridin is a highly fungistatic metabolic product of certain strains of Trichoderma viride. All viridin‐producing strains of this mould so far examined produce a yellow pigment abundantly in culture (Weindling's P series), which distinguishes them from the gliotoxin‐producing strains which in all recorded cases have been of the n on‐pigment‐forming type (Weindling's O and Q series).Cultural conditions affecting production of viridin in liquid media have been studied. A range of sugars and polysaccharides, and glycerol, can be utilized for the production of fungistatically active culture filtrates. Not all nitrogen sources which will support active growth are suitable for viridin production. Ammonium salts or peptone are quite suitable but nitrates are not. It is shown that media containing nitrates show a consistent tendency to develop a high pH during the growth of the mould, even where the medium is adjusted to pH 3‐5 initially. Viridin is known to decompose in aqueous solution at or near neutrality, and it is the characteristic pH drift of nitrate‐containing media which is probably responsible for their unsuitability for viridin production. There are minor differences in the nutrient requirements of the three strains of T. viride studied.The depth of the medium is of importance in determining the fungistatic activity of culture filtrates. On shallow layers (0.5 cm.) a peak of activity is usually reached about the sixth day of incubation at 250 C, after which activity falls off rapidly. There are marked differences in the drift of activity of culture filtrates of the three strains of T. viride studied; strain 213, which produces the most active culture filtrates, shows the most acute peak of activity. Deepening the medium has the effect not only of increasing the time taken to reach maximum fungistatic activity, and the degree of activity, but also leads to an absolute reduction in the amount of fungistatic material produced.Viridin may be isolated from active culture filtrates by extraction with chloroform, evaporation to dryness and recrystallization from ethyl alcohol, methyl alcohol or benzene. The crystals produced from each of these three solvents differ in form, but analyses of all agree with the formula C20 H16O6. Viridin is soluble in chloroform, hot ethyl alcohol and hot methyl alcohol; it is sparingly soluble in carbon disulphide and carbon tetrachloride, and almost insoluble in ether. It is optically active; for a i % solution in chloroform [x]19 D is ‐ 222°. An account is given of some of the chemical reactions of viridin. Viridin shows a marked degree of specificity in its fungistatic effects. Germination of conidia of Botrytis allii is prevented at 0‐006 pg./ml., whereas 6‐25/μg./ml. is needed to prevent germination of conidia of Penicillium expansum. Trichoderma viride conidia are insensitive to as much as 50/μg./ml. Higher concentrations are needed to be fungicidal; for Botrytis allii a concentration icoo times greater than the minimum needed to prevent germination is needed to give a complete kill by 2 hr. exposure. Viridin has little if any antibacterial action, and is therefore unique among all antibiotic mould products so far described, as all those previously described showing antifungal activity are also antibacterial. Aqueous solutions of viridin are not stable unless acid. At pH 29 half the activity of a solution is retained after 14 days at 250 C, at pH 58 all activity is lost in 1 day, and loss of activity is immediate at pH 8‐4. Aqueous solutions of viridin are inactivated by peptone and yeast extract, and by a number of pure chemical compounds likely to be present in such materials.