31 8 Journal of Agricultural Research voi. xm. no. 6 



Production of Apothecia in Pure Culture 



The cultures which are obtained from ascospores discharged on an agar 

 behave in early stages of development very differently from cultures 

 which have been obtained from mycelium in the leaf tissue. Mycelium 

 develops very slowly. Even when the agar surface has been abundantly 

 strewn with spores, mycelium does not become visible until after a week 

 or lo days. It grows almost wholly within the surface of the substratum 

 and soon unites to form a crust that varies in color with the nutrient 

 material furnished. Enormous numbers of conidia are produced that 

 sometimes cover the crust with a slime. If this crust covers the entire 

 surface of an agar slope, aerial mycelium is rarely developed in visible 

 amount. Such a culture appears very different from one developed from 

 mycelium from the host tissue. However, if a fragment of this crust is 

 cut out and placed on a fresh agar surface, mycelium v/ill emerge from 

 its edge; and in the course of a few v/eeks transfers can be made which 

 are indistinguishable from transfers from cultures obtained from host 

 tissue. 



This peculiar crust produced by mycelium from ascospores was not 

 carefully studied until the discovery had been made that one such cul- 

 ture was producing apothecia. Apothecia from this and other sources 

 were immediately used to start other cultures to determine the conditions 

 requisite for the development of the ascigerous stage. Since the supply 

 of ascospores was somewhat limited, and since the time required for the 

 formation of apothecia under the best of conditions found is from five to 

 eight weeks, it has not been possible to carry this part of the work to a 

 satisfactory conclusion. In no case have apothecia appeared in all the 

 cultures of a set that have been held under identical conditions. This 

 may be due in part to the fact that it is not possible to get two slopes 

 "seeded" with even approximately the same number of spores. Thus 

 far the ascogenous stage has been produced in 20 cultures. 



The best culture medium for this purpose appears to be oatmeal agar, 

 though apothecia have been produced on potato agar and alfalfa stems. 

 Spores may be discharged on a layer of clear-water agar in a petri dish, 

 where their number and distribution may be observed before transfer 

 with the substratum to the agar slope in the test tube; or they may be 

 discharged directly upon the agar slope. The latter method appears to 

 be somewhat better. 



With regard to temperature and light, by far the best success has been 

 attained in cultures exposed to weak light in a room which normally 

 maintains a temperature of about 21° C, but which at several times dur- 

 ing the growth of the cultures fell to 14° to 16° for several days. Con- 

 tinued lov/ temperature does not appear to favor the production of 

 apothecia. Cultures grow only slightly at 8°. Cultures held at constant 

 temperatures below 14° have shown no indication of producing apothecia 



