324 Journal of Agricultural Research voi. xiii, no. 6 



from an alfalfa field close beside the greenhouse; but during two winters 

 all plants in the greenhouse except those inoculated have remained free 

 from the disease. 



Ascospores for inoculation have been obtained from dead leaves from 

 the field, from apothecia on alfalfa leaves under conditions of pure cul- 

 ture, and from apothecia produced on culture media. The following 

 method of inoculation, which was first devised to secure the discharge of 

 ascospores from dead alfalfa leaves upon the leaf to be inoculated with 

 the least possible danger of transferring conidia or other spores, has been 

 used in many inoculations with this fungus. For this purpose a small 

 glass dish is made by cementing a heavy cover glass to one side of a glass 

 ring about 15 mm. in diameter and 6 mm. deep. In the bottom of the 

 dish thus formed is placed a fragment of dead leaf bearing apothecia 

 which appear mature. If dry leaves from the field are used, they should 

 be kept thoroughly wet for at least 12 hours before being used to obtain 

 the best discharge of spores. These leaf fragments remain firmly attached 

 to the bottom of the glass dish when kept wet. In order to determine 

 whether spores are actually being discharged and how abundantly, the 

 glass dish is then inverted on a thin layer of clear-water agar in a petri 

 dish. If, after an hour, very few or no spores are found on the agar, the 

 fragment is discarded and a new one substituted. The agar plates are 

 kept to ascertain whether the spores are viable. 



The dishes containing material that is discharging a suitable abundance 

 of spores is then placed over an alfalfa leaflet which is supported so that 

 the petiole is not greatly bent. The leaf is previously wet with a fine 

 spray, or rubbed slightly to cover it with a thin film of water, or some- 

 times it is sprayed after the spores are applied. After the dish has 

 been inverted over one leaflet for an hour or more, it is transferred to 

 another. The leaves thus inoculated are marked. It has been found 

 that apothecia on dead leaves can usually be depended upon to main- 

 tain a discharge of spores for at least 18 hours. The inoculated plant 

 is kept in a moist chamber for at least 12 hours after the last set of 

 leaves has been inoculated. 



Another method that has also been used consists of placing the material 

 from which ascospores are being discharged in the top of a bell jar over 

 plants which are well sprayed. This method has produced very abundant 

 infection on young plants, especially on vigorous seedlings; but with the 

 old plants infection is usually meager. Apparently, young tender 

 leaves are much more easily infected than older ones, although infection 

 may take place at any age. 



One of these inoculations with ascospores from dead leaves made on 

 November 4 and 5, 191 5, is summarized in Table II as the result appeared 

 on November 22. Several of the inoculated leaves had fallen at this 

 time and are not included in the results. 



