222 Journal of A gricultural Research voi. xvi. No. 8 



size of the spots varies from a pinhead up to 8 mm. in diameter at the 

 widest part, and the average diameter of the mature spot is 4 mm. 

 The most striking feature is the irregularly angular shape and the uneven 

 jagged outline (PI. 25-27). The center of the spot is tan or reddish 

 brown, with a darker thin border and often a suggestion of zonation which 

 is never conspicuous. A small, dark center is usually seen. In earlier 

 stages the spots are darker in color, almost black ^ at first, and are circular 

 or only slightly angular (PI. 25, A). The center of the spot becomes dry 

 and thin with age, paler in color until almost white, and may drop out, 

 leaving irregular holes which are sometimes so numerous with the conflu- 

 ence of spots that a skeleton leaf composed only of the viens remains. 

 Most of the tobacco is harv^ested before this stage is reached. No sharply 

 defined halo is present, but a narrow clear zone on the border is seen by 

 transmitted light. The tissue bordering the spots is yellowed, but this 

 diffuses gradually into the normal green. Usually the spots are more 

 numerous on one side of the midrib. 



In stained sections of fixed material the organism is found in great num- 

 bers throughout the shrunken tissue included in the spot. It is found 

 both within and betv/een the cells, and vacuolated cells are completely 



filled with it. 



INOCULATION EXPERIMENTS 



Proof of the pathogenecity of the organism isolated from leafspot 

 material was obtained through inoculations on seedling tobacco plants 

 (Warne variety). This work was carried on in the greenhouse during 

 the winter months. In all, some 150 plants were inoculated, and fully 

 96 per cent developed spots. All five isolations of the organism pro- 

 duced infection. Infection was readily obtained by atomizing the plants 

 with aqueous suspensions of the organism, by swabbing the leaves with 

 the cotton plug of bouillon cultures and by puncture with contaminated 

 needles. It was found necessary to place the plants in moist chambers 

 for 24 hours subsequent to inoculation. Some plants which were inocu- 

 lated and left in the open greenhouse failed to develop any spots, and 

 none of the many control plants became infected. No leaf injury is nec- 

 essary for infection. Apparently the organism gains entrance through the 

 stomata on either the upper or lower surface of the leaf. The inoculum 

 has been recovered in pure culture a number of times, and these re- 

 coveries have been used for reinfections. Secondary infections have never 

 developed, but this is a common experience with plant pathogens under 

 the dry atmospheric conditions of the greenhouse. 



As many as 200 spots were obtained on a single leaf through inoculation 

 with the atomizer, but the average ran much lower than this. Still 

 heavier infections were obtained by swabbing with contaminated cotton 

 plugs. The spots obtained were typical of the spots seen in the field, 

 but were smaller. They averaged about 1.5 mm. in diameter, and 



' One farmer stated that his first impression of the spot was that someone had spattered ink on the leaves. 



