In vitro growth of wheat and flax rust fungi on complex and chemically defined media

Abstract

Growth from uredospores seeded in axenic culture is described for several races of Puccinia graminis Pers. f. sp. tritici (Erikss. and Henn.) and race 3 of Melampsora lini (Ehrenb.) Lév. on complex media containing peptone, yeast extract, and bovine serum albumin (BSA); and for an Australian isolate of Puccinia graminis, race 126-ANZ 6,7, and Melampsora lini, race 3, on chemically defined, liquid media.Of six North American isolates of Puccinia graminis only race 38 formed colonies approaching those of race 126-ANZ 6,7 in final size and general morphology on complex media. 5′AMP had no effect on the growth of 126-ANZ 6,7, but cyclic AMP inhibited growth after uredospore germination. Good growth and sporulation were obtained with 126-ANZ 6,7, but not with the other isolates tested, using a new, chemically defined liquid medium, sterilized by millipore filtration, and containing glucose, Czapek's minerals plus micronutrients, Ca2+, glucose and aspartic acid, glutathione, and cysteine. Uredospores produced in culture reinfected exposed mesophyll tissue, but not intact seedling leaves of wheat.Highly reproducible growth and sporulation of Melampsora lini, race 3, were obtained routinely on a solid medium containing Difco-Bacto agar, sucrose, Knop's minerals, micronutrients, yeast extract, peptone, and BSA. Vegetative cultures, capable of reinfecting the cut ends of surface-sterilized flax cotyledons, could be maintained indefinitely by subdivision before sporulation and transfer to the same medium minus BSA. Evidence is presented that BSA stimulated the development of colonies and the formation of uredospores. The mode of action of BSA is unknown, but it could not be replaced by putrescine.A new chemically defined, liquid medium containing sucrose, Knop's mineral salts, micronutrients, aspartic (or glutamic) acid, and cysteine supported the growth of colonies of Melampsora lini in a highly reproducible manner. The formation of uredospores and teliospores by these colonies was controlled by (a) the level of Ca2+ (as Ca(NO3)2∙4H2O), (b) the concentration of aspartic acid, and (c) the number of colonies per flask. At inoculum levels giving 40 to 60 colonies per flask, in media containing 8.5 mM Ca+ and 45 mM aspartic acid, uredospore formation occurred in 60 to 70% of the colonies. A decrease in the Ca2+ level to 4.25 mM, or a decrease in aspartic acid to 22.5 mM, or adjustment of the inoculum level to give about 10 colonies per flask each resulted in only infrequent sporulation. The uredospores produced in vitro infected intact, 1-week-old flax cotyledons in a normal manner.