Fig-. 6. Young corn plants severely injured by Ustilago maydis and prostrate on the ground, hence subject to 

 rapid disintegration (Stakman and Christensen, 311). 



the same whether inoculated with local or nonlocal 

 smut collections; hence, physiologic races do not appear 

 responsible (57). There is some evidence that certain 

 morphological characters, i.e., lack of ligules and tight- 

 ness of husk, may be involved in some cases (157, 193 I. 



Isolation of U. maydis. — Cultures can readily be 

 obtained by streaking chlamydospores on 1-2% potato- 

 dextrose agar or any other solid nutrient agar favor- 

 able to the growth of the fungus. If the smut gall is 

 infested with other microorganisms, it is desirable to 

 soak the chlamydospores for 20-50 hr in 1% copper 

 sulfate before plating them on nutrient agar. 



The method of isolating single chlamydospores or 

 sets of sporidia is simple and relatively easy with a 

 micromanipulator (55, 79, 122). A good method is as 

 follows: a thin fiat film of clear nutrient medium con- 

 taining 2 ( ' f agar is placed on the surface of a cover- 

 glass. With a dry glass needle attached to a micro- 

 manipulator, a chlamydospore is picked up from 

 another dry cover-glass previously dusted with dry 

 chlamydospores. Then, the spore is transferred to a 

 marked spot on the agar film. This cover-glass is in- 

 verted over a Van Tiegham cell in a moist chamber 

 such as a petri plate. 



After the chlamydospore germinates, the sporidia can 

 be isolated directly from the promycelium or an indi- 

 vidual sporidium can be drawn away from the promy- 

 celium, along the surface of the agar, until it is some 

 distance from the promycelium and other sporidia. The 

 needle then is pushed into the agar a couple of times to 

 create a slight depression in which the sporidium might 

 bud; and, also, to mark the location of the sporidium 

 (55). The isolated sporidium is allowed to bud for 

 24-36 hr, depending on the kind of medium: the 

 temperature; and, to some extent, on the rapidity of 



growth of the particular line of smut. From these small 

 colonies, sporidia are easily removed with the needle of 

 a micromanipulator to another sterile drop of agar. 

 From the colonies produced on the new sets of cover- 

 glasses, transfers can usually be made to agar slants 

 within about 2 days. If the original isolated sporidia 

 are properly spaced on the agar films, they can be 

 transferred directly to agar slants (55). The sporidia 

 are normally numbered according to their position on 

 the promycelium. The sporidium at the tip of the 

 promycelium is usually designated no. 1 and the one 

 nearest the spore, no. 4. 



When a large population of subcultures is desired, 

 a suspension of sporidia is sprayed or sown on the dry 

 surface of nutrient agar plates. The excess liquid can 

 be removed and kept off by using a clay drying cover. 

 This keeps the small colonies from flowing together 

 during multiplication of the sporidia (203). 



In order to reisolate sporidia from mycelium in the 

 host, the following procedure is suggested. Four-6 days 

 after inoculation, leaves of corn plants on which there 

 is evidence of good infection, are cut off and disin- 

 fected. They are dipped in 70% alcohol, immersed in 

 mercuric bichloride. 1:1,000 for 2-i min. dipped in 

 alcohol again, then rinsed in sterile distilled water or, 

 better still, in 1 or 2% solution of sodium hypo- 

 chlorite. Following this treatment, the leaves are 

 incubated in a sterile moist chamber. It usually 

 requires 3-4 days for aerial sporidia to develop on the 

 infected parts. The individual sporidia can be isolated 

 in the usual manner. 



Spores. — Morphology of spores. — U. maydis is a 

 good example of the wide diversity that can exist in the 

 size and shape of spores within a smut species and illus- 

 trates the need for an adequate sample of spores in 



12 



