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The axenic culture of wheat and flax rust fungi Bose, Amitava

Abstract

Rust fungi belonging to the order Uredinales have usually been considered the classical examples of obligate parasites amongst plant pathogenic fungi. The development of our knowledge of the metabolism, nutrition and physiology of the rust fungi has been restricted because of our inability to grow these fungi in axenic culture. In the past the physiological and biochemical investigations on rust fungi perse have been limited to studies of the germination and development of germ tubes. The cultivation invitro of Puccinia graminis tritici (Erikss . and .Henri.), Australian race ANZ 126-6,7, by Williams et al. (1966, 1967), together with the report of Turel (1969) on the axenic culture of Melampsora lini (Ehrenb) Lev, race 3 promoted research on culturing rust fungi on artificial medium. An artificial medium containing 3% glucose, Czapek's Mineral salts, 0.1% Evan's peptone, plus defatted BSA supported growth and sporulation of Puccinia graminis tritici race ANZ 126-6,7. Typical pigmented uredospores and teliospores were formed after 6-3 weeks growth. The uredospores were capable of infecting the mesophyll of wheat leaves exposed by stripping back the lower epidermis. Scanning electron microscopy revealed the presence of a coating of unknown chemical composition around the uredospores developed in vitro and not observed on uredospores grown on wheat leaves. Two different strains of Melampsoralini (Ehrenb) Lev were grown on solid media containing 4% sucrose, modified Knop's tissue culture macro nutrients, Berthelot's micro nutrients, yeast extract and peptone. The mycelium was generally binucleate. Spore-like structures were recorded in the stroma which resembled uredospores and teliospores . Addition of 1% defatted BSA to the medium described above greatly increased the frequency of establishment off lax rust colonies. A defined liquid medium, containing Czapek's minerals y Ca++, glucose, as particacid, glutathione and cysteine, and inoculated with uncontaminated uredospores, supported good vegetative growth and sporulation of wheat stem rust (Puccini agraminis f. sp. tritici race ANZ 1 2 6 - 6 , 7). Of six North American races of wheat stem rust fungus tested, only three grew vegetatively on artificial medium. Finally a chemically defined liquid medium containing sucrose , Knop's mineral salts, micro nutrients, aspartic (or glutamic) acid and cysteine supported the growth of vegetative colonies of Melampsora lini race 3 from uncontaminated uredospores in a highly reproducible manner. The formation of uredospores and telio spores of these colonies in the liquid medium was controlled by the level of Ca++ (as Ca(NO₃)₂) and the number of colonies per flask. With 60-70 colonies per flask, uredospore formation occurred on 60 to 70% of the colonies at a Ca++ level 8. 5 mM. A decrease in the Ca++ level to 4.5 mM or colony frequency to 10 per flask resulted in only in frequent sporulation vitro infected in tactcotyledons result with flax rust represents ability to control the growth of parasite in axenic culture. The uredospores produced in a normal manner. This a substantial advance in our this important obligate parasite in axenic culture.

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