38 BULLETIN 934, U. S. DEPARTMENT OF AGRICULTURE. 



readily at certain times of the year was done before the development of pure- 

 culture methods. Water cultures kept in the dark and in the light, at constant 

 and at varying temperatures, with nutrient substrata consisting of steamed 

 or outoclaved fragments of potato, carrot, sweet potato, turnip, sugar beet, coru 

 meal or rice, nutrient agar, sugar-beet seedlings, and insects have all produced 

 only sexual fruiting bodies and chlamydospores (the so-called conidia). 



(3) The successful cross-inoculations, those which Edson (38) used on sugar- 

 beet strains and the writer had found parasitic on pine and had used on pine, 

 strains which Hawkins had found parasitic on potato tubers and Edson on 

 sugar beets, confirm the work of Hofmann (77) in indicating that the .Pythium 

 which causes the damping-off pine is a parasite on entirely unrelated species 

 of host plants, a commonly recognized characteristic of Pythium debaryanum. 



The organism is easily isolated from recently damped-off conifer- 

 ous seedlings or from soil direct by placing the seedlings or a lump 

 of soil at the edge of a Petri dish of solidified prune agar and transfer- 

 ring to tubes mycelium from the advancing edge of the resulting 

 growth. It has been found in or obtained from damped-off conifers in 

 California, Kansas, Nebraska, Minnesota, and the District of Columbia, 

 as well as in cultures made by Mr. Glenn G. Hahn in Michigan. Picea 

 engelmanni, P. sitche?isis, Tsuga mertensiana, Pinus nigra austriaca, 

 and P^eudotsuga taxi folia are the coniferous hosts from which cul- 

 tures of Pythium. debaryanum have been obtained. It has been iso- 

 lated directly from soil not only in coniferous seed beds but from 

 open grassland in California not adjacent to any seed bed or culti- 

 vated crop. Unless Mucor is abundant, Pythium is commonly ob- 

 tained in apparently pure condition on the first transfer from the 

 plate, as prune agar appears unfavorable for most bacterial growth 

 while allowing rapid spread of the Pythium. On media made from 

 prunes which taste sweet and with a total gross weight of not more 

 than 40 or 50 grams per liter of medium, the Pythium will make a 

 rapid growth, often extending radially 1 mm. per hour at tempera- 

 tures in the neighborhood of 22° C. and produce both chlamydospores 

 and oospores. A less valuable medium for isolation work, but more 

 convenient for subcultures than any other which has been tested, is 

 autoclaved corn-meal agar. The growth is not luxuriant, but spores 

 are always formed and the cultures seem to be as long lived as those 

 on any other medium, retransfer being rarely necessary more often 

 than twice a year. Much stronger growth and more abundant fruit- 

 ing is obtained on such media as sugar-beet or rice-stem agar, but 

 the leathery surface of the culture on such media makes transferring 

 difficult. On rice grains, corn-meal mush, beef agar, and on corn- 

 meal agar plus 2 per cent dextose or sucrose no spores are formed 

 and the cultures are short lived, though growth is heavy and on 

 the last-named medium extremely rapid. On agar containing the 

 juice from sour prunes or on corn-meal agar prepared without sub- 

 jecting it to the high temperature of the autoclave, both growth and 

 fruiting have been very poor or even lacking. 



