Apr. 29. 1918 Stemphylium Leaf spot of Cucumbers 297 



scope and a few spores transferred by means of a sterile, moistened scalpel 

 to string-bean agar. Dilution plate cultures were then made. As soon 

 as the spores had germinated, as shown by examination under a micro- 

 scope, single spores were transferred to other agar plates. When these 

 cultures produced spores, single-spore isolations were again made to 

 make doubly sure they were free from contamination. In a few cases 

 the fungus was isolated by the tissue-culture method from leaves. In- 

 fected leaves were sterilized by washing for one-half to three minutes 

 in a I to 1,000 solution of mercuric chlorid or in 95 per cent alcohol. 

 They were then thoroughly washed with distilled water and plated in 

 string-bean or potato agar. In some cases these cultures were overrun 

 b)7 various mold fungi, but in others a fungus identical in all respects 

 with that obtained from the spore dilutions was isolated. The fungus 

 was isolated from material collected in 191 6 at Lapaz and Hamlet, Ind. 

 These isolations were labeled as strains i and 2, respectively. A tissue 

 culture isolation from Lapaz was labeled "strain 3." Cultures from 

 two of the mold fungi were retained and used for later inoculations. 



INOCULATION EXPERIMENTS 



Inoculation tests were made with cultures from the strains mentioned 

 above as well as with spores taken directly from diseased cucumber 

 leaves. All strains, as well as the fresh spores, developed abundant 

 infection. Inoculations made with the other fungi isolated from the 

 diseased spots failed to develop infection. Most of the inoculations 

 were made in the greenhouse, only one series being made in the field. 

 In both cases the inoculations were successful. 



One series of inoculations made on September 13, 1916, will be de- 

 scribed in detail as being typical of the manner in which all were made. 

 Spores of strains 1,2, and 3 were taken from string-bean-agar cultures 

 15 days old. Contemporaneous germination tests on slides showed that 

 the spores were viable. Inoculations were made with suspensions of 

 these spores in distilled water, tap water, and beef bouillon. The spores 

 were applied to both upper and lower surfaces of the leaves by spraying 

 the suspension from an atomizer or by dropping it from a pipette. In 

 spraying with the atomizer some of the spores doubtless occasionally 

 reached both surfaces. Four plants were used with each strain for 

 each different method of inoculation, 12 plants being sprayed with 

 water and 6 with bouillon as controls. One-half of the plants were 

 left in the dry air of the greenhouse. The others were placed for 24 

 hours in a glass culture chamber so arranged that fresh air was ad- 

 mitted from below, and after this period they were also kept in the 

 greenhouse. The results on September 17, four da3^s later, are sum- 

 marized in Table I. 



