246 Proceedings of Indiana Academy of Science 



One of these is the toxicity of the water. At this laboratory, no detri- 

 mental effect has been obtained from the use of tap water. Melhus and 

 Durrell (1919), however, found tap water toxic to urediniospores of 

 Puccinia coronata at Ames, Iowa, and used distilled water in their inocu- 

 lations. Another factor of importance is the submersion of the spores. 

 Aeciospores and urediniospores of Puccinia triticina give little or no 

 germination if submerged and the teliospores, if they germinate at all, 

 produce long tubes without forming basidia. This has been noted by a 

 number of investigators (Melhus & Dui'rell, 1919; Melhus, Durrell & 

 Kirby, 1920; and Doran, 1921). 



5. Inoculation — Urediniospores. The inoculation method used de- 

 pends upon the type of investigation being conducted. Where either a 

 few plants are being inoculated, or the amount of inoculum is small, or 

 where different strains of rust are being studied, necessitating carefully 

 controlled conditions, each plant is inoculated with spores applied with 

 a scalpel. The plants are first wet by atomizing with a De Vilbiss 

 atomizer as recommended by Melhus (1912). In the case of the cereals, 

 a waxy coating on the leaves prevents water from adhering. This is 

 overcome by gently drawing the leaves between wet fingers, as recom- 

 mended by Melchers (1915), until water adheres. 



Spores are removed from the rusted plants with a straight-edged 

 scalpel and spread on the wetted leaves. It has not been found neces- 

 sary to trim the inoculated plants of all leaves except those inoculated 

 and no difficulty has been found in interpreting the results. Inocula- 

 tions with the leafrusts of wheat, rye, barley, and corn give practically 

 as good results when the spores ai-e placed on one surface of the leaf as 

 on the other. This enables rapid sowing of spores, as a number of 

 plants may be inoculated by holding the leaves by the tips so that they 

 are in one layer and covering them all with spores at the same time. 

 Hungerford and Owens (192.3) state that in the case of Pi(cci}tia (jhtvi- 

 ariun good infection is obtained only when spores are sown on the upper 

 side of the wheat leaf. Melchers (1915) recommends the sowing of 

 Puccinia graviinis on the lower side of the leaf. Where more than one 

 culture of the same rust is being studied the usual precautions of steril- 

 izing the instruments used must be taken. 



Where plenty of viable urediniospores are available and only one 

 culture of a species is under greenhouse investigation, dusting with 

 spores has been found the most effective method of inoculating large 

 numbers of plants in a manner similar to that recommended by Fromme 

 (1913). The plants are first atomized and then heavily rusted plants 

 are shaken over them, after which they are placed in a moist incubation 

 chamber. By this method the urediniospores are well scattered and the 

 exposed plant parts usually are fairly uniformly inoculated. Whei'e this 

 method can be used, it has the advantage that a large number of plants 

 can be inoculated quickly. 



Aeciospores. Similar methods ai'e used with aeciospores. There 

 are several modifications, however, which have been found very service- 

 able. As aeciospores are catenulate, the lower spores being immature 

 when the upper are ripe, scraping the aecia off with a scalpel removes 

 many immature spores. When aecia have been produced in the green- 



