STUDIES OF TWO POLYCENTRIC CHYTRIDS IN PURE CULTURE

GOLDSTEIN, SOLOMON. (Yale U., New Haven, Conn.) Studies of two polycentric chytrids in pure culture. Amer. Jour. Bot. 48(4): 294-298. Illus. 1961.-A study was made of some of the chemical and physical factors affecting the growth of Nowakowskiella elegans Schroeter and N. ramosa Butler in pure culture. Maximal harvests of the former were obtained at 150C. and those of the latter fungus at 200C. Media buffered at pH 6.4 favored their growth; light had no effect upon yields; and harvests increased markedly in response to shaking. Both fungi required an exogenous supply of thiamin with maximum yields obtained upon incorporating 10 /g./l. of the vitamin into a thiamin-deficient medium. Inorganic nitrogen salts and several organic nitrogen compounds were readily metabolized by these water molds. The use of xylose, glucose, or mannose as the sole source of carbon gave optimal yields of both organisms and neither utilized a variety of sugar acids and alcohols. MEMBERS of the genus Nowakowskiella constitute a morphologically well-defined group of cosmopolitan cellulose-decomposing chytrids. Because of the polycentric organization of their thalli, it has been suggested (Roberts, 1948) that they may represent possible transition forms between the monocentric chytrids and those Phycomycetes that possess a truly hyphal structure. Although the morphology of chytrids has received considerable study, investigations of their physiology have been limited in number and scope. Haskins (1939) noted the ability of several chytrids, Nowakowskiella elegans among them, to develop on bits of leaf tissue devoid of protoplasmic contents. Upon replacing such tissue with a variety of cellulosic materials including cellophane, he found that the organisms continued to thrive, whereas contamination in the cultures decreased. Couch (1939) stimulated physiological studies of chytrids with the publication of his techniques for the isolation and growth of a number of these fungi in pure culture. He also recorded his inability to bring N. elegans, which had been maintained on boiled filter paper in gross cultures, into pure culture on that substrate. This finding, however, has been criticized by Stanier (1942) on the grounds that the organism was not provided with a source of nitrogen. Couch suggested that the presence of bacteria might be required for the growth of some chytrids on cellulosic materials. Whiffen (1941), employing a cellu1 Received for publication June 13, 1960. This paper is a portion of a dissertation submitted to the Horace H. Rackham School of Graduate Studies of the University of Michigan in partial fulfillment of the requirements for the degree of Doctor of Philosophy. I want to thank Professor F. K. Sparrow, Jr. for his help and encouragement throughout the course of this work. I wish also to thank Professor A. Geoffrey Norman for his many helpful suggestions and for placing the facilities of the Plant Nutrition Laboratory at my disposal. I am indebted to Dr. Melvin S. Fuller of Brown University for the culture of Nowakowskiella elegans. Grateful acknowledgement is made of the financial support given by the Department of Botany of the University of Michigan. Contribution from the Department of Botany of the University of Michigan No. 1120. lose-ammonium nitrate medium, demonstrated that N. elegans and several other chytrids were capable of decomposing cellulose directly and that they were not dependent upon hydrolytic products formed by microbial contaminants. The present investigation was begun in order to extend our knowledge of polycentric chytrids and to develop techniques that would make feasible studies of their intermediary metabolism. MATERIALS AND METHODS.-The fungi employed in this study, Nowakowskiella elegans Schroeter and N. ramosa Butler, members of the Megachytriaceae, are operculate, polycentric chytrids which possess non-septate turbinate cells. These 2 species have been distinguished morphologically from each other on the basis that N. elegans never forms pseudoparenchymatous tissue and rarely, if ever, produces resting spores, whereas N. ramosa exhibits both of these features (Sparrow, 1960). A culture of N. elegans, which had been isolated from a sample of California soil, was obtained from Dr. Melvin S. Fuller. The strain of N. ramosa was brought into pure culture from vegetable debris gathered from along the shore of Third Sister Lake, Washtenaw County, Michigan. Because of the problems attendant upon the isolation of chytrids (Emerson, 1950), it seems worthwhile to recount the techniques employed to accomplish this. A piece of uncoated cellophane upon which the fungus was growing was removed from a gross culture, washed with sterile distilled water, blotted with sterile filter paper, and placed on the dry surface of cellulose agar medium in a Petri dish. The cellulose had been dissolved with sulfuric acid and precipitated according to the directions given by Scales (1916). The culture was examined daily with the aid of a binocular dissecting microscope and, after a 10-day incubation period, sporangia were observed to have formed on the surface of the agar. Portions of the agar bearing sporangia and rhizomycelium formed at some distance from the original point of inoculation were cut out with a