The Nature of Cold - induced Dormancy in Urediospores of Puccinia graminis tritici Received for publication

When air-dry urediospores of the wheat stem rust, Puccinia graminis f. sp. tritici, are exposed to temperatures below freezing, their germinability is markedly reduced, even after prolonged thawing at room temperature. Germinability is fully restored by a brief heat-shock or by vapor phase hydration. We have found that this "cold dormancy" cannot be reversed once the spores contact liquid water. Enhanced loss of metabolites occurs immediately upon suspension of cold-dormant urediospores in liquid without a prior heat-shock. Such leakage is two to three times greater than from untreated or heat. shocked cold-dormant spores and accounts for up to 70% of the soluble pool of metabolites normally present in germinating uredio8pores. Respiratory activity of cold-dormant urediospores declines rapidly during incubation in liquid. Incorporation of isotopic carbon into cold-dormant urediospores is only a fraction of that of untreated or heat-activated spores. Thus, cold shock transforms the spores into a state of supersensitivity to liquid water, which is reversed by heat-shock or slow hydration by vapor phase equilibration. The primary cause of damage to cold-dormant cells exposed to liquid water appears to be irreversible permeability damage, followed by metabolic injury. The urediospores of rust fungi are short lived, and so these organisms are maintained by periodic infection of host plants. Therefore, were it possible to preserve such spores in a viable condition, they would be more readily available for use in plant-breeding programs directed against these pathogens, as well as for physiological and biochemical studies. In fact, survival of urediospores of rust fungi by storage in liquid nitrogen has been demonstrated (3, 7, 18). However, it has been shown that their germinability after freezing critically depends on the method of revival (3, 13). Thus, when air-dry urediospores of Puccinia graminis f. sp. tritici Erikss. and Henn., races 15 and 56, are cooled below freezing to -196 C, germination is markedly reduced. Inasmuch as germinability is restored by a heat-shock (14) and by vapor phase hydration (3), these authors concluded that freezing induces a reversible cold dormancy in urediospores. Recently, we demonstrated (34) that there are no obvious ultrastructural differences between frozen and untreated urediospores before suspension in water. However, grievous ultrastructural changes developed during rehydration of cold-dormant spores in a liquid medium. In this paper we shall present evidence showing that cold dormancy is, in fact, a condition of supersensitivity of frozen spores to rapid hydration by liquid water, resulting in irreversible damage to the permeability of the spores. MATERIALS AND METHODS Urediospores of P. graminis pers. f. sp. tritici Erikss. and Henn., race 56, were used throughout. These were produced on Triticum aestivum L., var. 'Baart', C. I. 1967, in a controlled environment chamber under a photoperiod of 16 hr, 200 ft-c, at 22 C, and 8 hr of darkness at 20 C. The spores had a moisture content of 1 1 to 16% as determined by drying at 60 C for 48 hr. Spores generally were used the same day as collected. Production of "4C-Labeled Urediospores. Isotopically labeled urediospores were produced on rust-infected wheat plants allowed to photosynthesize 14CO2 in a sealed glass chamber. Five 4-inch pots, each containing 50 to 70 wheat plants, about 6 inches tall and at the fleck stage of pustule development, were arranged on a Plexiglas base. The plants were covered with a glass chamber measuring 151/2 X 151⁄22 X 16 inches which was sealed with a caulking compound. The whole assembly was placed in a controlled environment chamber operated as described above. Radioactive CO2 was produced by reacting Ba14CO. with lactic acid in a test tube connected to the glass chamber through a side port fitted with a valve. Inflow and mixing of 'CO. was assured by creating a slight negative pressure in the chamber before the gas was introduced. After the reaction was over, water was introduced into the tube to displace the residual 4CO2, and then air was allowed to bubble into the chamber until equilibrium was reached. Radioactivity inside the chamber was monitored by withdrawal of gas at regular intervals with a gas-tight syringe through a vent. The gas was dissolved in Hyamine 10-X (Packard Instrument Co., Downers Grove, Ill.) in a rubber-stoppered scintillation vial and was counted in a toluene-based scintillation fluid which will be described later. In our experiments, 10 to 20 ,uc of "CO2 were introduced into the chamber on alternate days. Urediospores were collected every 3rd or 4th day by careful dislodgment from the leaves into a cardboard box lined with aluminum foil. The total yield of spores from a single experiment lasting 10 to 12 days ranged from 1.5 to 2.0 g (fresh weight). Before use the spores were sieved to remove debris, then thoroughly mixed, and dried over anhydrous calcium chloride in a desiccator to approximately 15% water content. These germinated to the extent of about 60 to 80%. Induction and Reversal of Cold Dormancy. Approximately 10 to 40 mg of air-dried urediospores were sealed in Pyrex tubes, 15 cm long and 6 mm wide (inner diameter), and cold