seems likely, however, that inoculation with chlamydo- 

 spores or sporidia might prove valuable in a region 

 where U. maydis is not particularly common. 



Injection of spores. — In 1900. Arthur and Stuart 

 i 5 i injected cultures of smut into seedlings of corn by 

 means of glass tubes. Now. the inoculation of corn 

 plants, especially seedlings, by means of a hypodermic 

 syringe is a common method (55. 118. 3141. Inocula- 

 tion of seedlings is particularly valuable for testing for 

 sexual compatibility among haploid lines, for testing 

 stability of virulence, and also on physiology of para- 

 sitism. The method also has been used to inoculate 

 older plants, which are more useful than seedlings in 

 studying physiologic specialization in U. maydis and 

 the nature of resistance to smut (311) (Fig. 8). 



The sporidial inoculum can be increased in test tubes 

 or flasks containing about 1% sucrose or a similar 

 carbon source, plus a small amount of potato or carrot 

 extract. Many other substrates can be used with equal 

 success. The organism is allowed to grow 1-2 weeks 

 before inoculation. Just before inoculation, the culture 

 is diluted 30-50 or more times its volume with water. 

 Whenever the inoculations are to be made with 2 or 

 more haploid lines, they are thoroughly mixed just 

 preceding injection into the host. 



Partial vacuum. — Wilkinson and Kent (358) intro- 

 duced a partial vacuum method for inoculating corn 

 with smut. Their technique was patterned somewhat 

 after the method used by Moore (230) for inoculating 

 barley and wheat flowers with loose smut. The inocu- 

 lum was dropped into the spiral whorl of seedlings. 

 Then, the plants were subjected to reduced pressure — 

 evacuation to 70 cm mercury — for 15 min. Then, on 

 the release of pressure, the inoculum was forced into 

 the meristematic region of the leaf spiral. About S5% 

 of the treated seedlings developed smut. 



Wallin (348 ) wounded seeds and then inoculated 

 them by covering them with a suspension of inoculum 

 and subjecting them to partial vacuum. Apparently, this 

 method is not very effective, at least it is not in 

 common use. 



In 1953. Rowell and DeVay (281) again modified 

 the partial vacuum method. Their method consisted of 

 germinating surface-disinfected corn seed under aseptic 

 conditions until the coleoptile was about 1 cm long. 

 Then, the tip of the coleoptile was cut off to expose 

 the plumule and the meristematic area. The seedlings 

 then were placed in tubes and covered with the sus- 

 pension of sporidia or chlamydospores to be tested. 

 After that, they were subjected to a partial vacuum for 

 5 min. The seedlings then were planted in sterile soil 

 or Yermiculite. This method has proved rapid and 

 efficient for testing compatibility of lines and for 

 making genetic studies of the pathogen. In 2 years. 

 Rowell and DeVay (281) made more than 17.000 tests 

 for pathogenicity and sexual compatibility, a good in- 

 dication of its efficiency. It is not a desirable method 

 for testing lines of corn for resistance, because young 

 seedlings are very susceptible and often killed by the 

 smut fungus (157, 281). 



Whorl inoculation. — Brefeld (34) used successfully 

 the technique of pouring a suspension of sporidia into 

 the leaf whorl of a corn plant. Since then, this method 

 has been used extensively, but in many cases with 



negative or poor results (5, 263, 351). Experiments 

 indicate that inoculum does not readily reach the apical 

 meristem because it is tightly enclosed by surrounding 

 leaves. If the leaves do not too tightly enclose the 

 meristem. it can be bathed in inoculum more readily. 

 Lines and varieties of corn differ in this characteristic. 



Davis (72) found that the percentage of smut- 

 infected plants was greatly increased when a surface 

 tension depressant such as fish-oil soap was added to 

 inoculum before inoculation. The purpose of the sur- 

 factant was to carry the inoculum down through the 

 spiral whorl to make direct contact with the meriste- 

 matic tissue. Dickson and Bowman (80) obtained good 

 results using this method, whereas Walter (351) did 

 not. Wilkinson and Kent (358) concluded that tri- 

 ethanolamine oleate was a better depressant than fish- 

 oil soap as it gave a higher percentage of infection. In 

 order to get consistent results, the authors emphasized 

 the importance of using a nontoxic surfactant which is 

 also stable and uniform in its constitution. 



Spiral loosening. — It is well established that the 

 injection of smut inoculum into the growing tissue 

 nearly always results in smut infection. Even forcing 

 dry chlamydospores into the spiral will induce severe 

 smut infection (351). Just how and when most of the 

 inoculum reaches the meristematic tissue of corn is not 

 known. In nature, the presence of inoculum apparently 

 is not the limiting factor because the injection of only 

 sterile water or broth into the leaf spiral when the 

 plants are 30-50 cm high will greatly increase the per- 

 centage of smut (351). The water presumably makes 

 a connection between the inoculum in the upper leaf 

 spiral and susceptible tissue in the apical leaf roll. 



Based on the above information. Walter (351) sim- 

 ulated the spiral-loosening effect of the wind by rolling 

 the leaf spirals between the palms of his hands during 

 or immediately following rain. This treatment greatly 

 increased the percentage of smutty plants, especially if 

 the plants were treated after they were about 30 cm 

 high and before they entered the early boot stage. 

 Walter concluded that heavy wind and rain storms 

 caused twisting and bending of plants, bringing the 

 smut inoculum down to the susceptible tissues in the 

 lower part of the leaf whorl. 



Infection. — Until about 1900. it was generally 

 assumed that U. maydis infected young seedlings in the 

 manner of other cereal smuts. Even Brefeld (34 ) held 

 this view as late as 1883. Kiihn (192). in 1874. claimed 

 that he observed the penetration of a germ tube of 

 U. maydis into the root-node of a corn seedling. It was 

 Brefeld who from 18S3 to 1895 proved that all tender 

 succulent parts of a corn plant above the ground line 

 were susceptible to infection. He established beyond 

 doubt that the place and type of infection were dis- 

 tinctly different from those of other cereal smuts. 



Local infection. — Brefeld (34) induced infection by 

 spraying the growing plants with a suspension of spo- 

 ridia and by dropping them into the leaf whorl of young 

 seedlings. He proved that the galls were the result of 

 local infection and that the corn plant continued to be 

 susceptible to new infection as long as there were 

 succulent and meristematic tissues. He also observed 

 penetration of the smut fungus into many different 

 organs of the plant. 



16 



