A STUDY OF A NEW SPECIES OF SPONGIOCHLORIS INTRODUCED INTO STERILE SOIL

A new species of Spongiochloris, S. typica, was isolated from a Connecticut soil. It can be distinguished from other species by the presence of many pyrenoids; by the cell size of light-grown cells (up to 80), usually with thin walls; and by the vacuolate older cells. Zoospores present favorable material for observation of withdrawal of flagella upon quiescence. The organism was reintroduced into sterile soil, the soil was then air-dried, and the viability of the organism in soil was measured during 1 year. Each month, a 0.5-g sample of soil was heated at 100 C for 1 hr prior to incubation in basal medium; a control sample was incubated, along with the treated soil, in continuous illumination. By means of 10-fold dilutions of such cultures, it was determined that more than 10,000 cells per 0.5 g of soil survived heat pretreatment and more than 100,000 cells per 0.5 g were viable in the desiccated soil after 1 year. STARR (1955) described the genus Spongiochloris, distinguishing it from other unicellular, zoospore-producing chlorococcacean algae on the basis of plastid type, presence of pyrenoids, and type of zoospore produced. Two species were described at that time, S. spongiosa and S. excentrica. MXIore recently Deason and Bold (1960) and Chantanachat and Bold (1962) characterized 3 additional species. All can be distinguished easily by means of both morphological and physiological attributes (Chantanachat and Bold, 1962). Survival of algal cells in dry soil, resistance to desiccation, and the morphology of cells in dry soils have been studied frequently (Bristol 1920, 1927; Fritsch, 1922; Fritsch and Haines, 1923). Trainor (1962) demonstrated that some algae in dry soils were extremely resistant to high temperatures. A number of organisms survived heat pretreatment at 100 C for 1 hr, and at least 1 organism in each of 2 different soils was viable after 1 hr at 140 C. The present study was undertaken in order to investigate the possibility of reintroducing an organism into sterile soil, to explore further its resistance to both airand oven-drying, as well as initiate a study of the role of this alga in soil. The organism in question is new to science and will be described. It was initially chosen because it can grow well over a wide temperature range and is also capable of growth in both light and darkness. MATERIALS AND METHODS-The alga described in this paper was isolated from a soil sample collected from a cornfield in Storrs, Connecticut, in October, 1958. In the initial isolation procedure, 0.5 g of soil was placed in a sterile 8-oz soft-glass bottle which contained 50 ml of basal medium, IN I Received for publication May 2, 1963. This investigation was supported by N S F grant G16106. (Trainor, 1958). This bottle was incubated under continuous illumination at 26 i 1 C. Although presence of algae was noted within a week, the organism was not isolated until the 6th week. Three other organisms, not discussed in this paper, also were isolated. The organism was established in axenic culture by using the standard techniques of repeated streaking and isolation of colonies from agar p ates. All experiments, other than initial incubation, were conducted at 24 -4 1 C. When continuous illumination was used, the intensity of fluorescent illumination was 400 ft-c. Slower growth of the organism for maintenance of stock cultures, as well as for observation under a more "natural" light cycle, was achieved under 12-hr diurnal illumination at an intensity of 200 ft-c. The organism was grown routinely in IN medium (Trainor, 1958). For comparison with other known species, soil extract (Pringsheim, 1949) and standard bacteriological nutrient broth were used. All ages of cells, from recently released zoospores to cells from 6-month-old cultures, were observed microscopically. These cultures were grown under both continuous and diurnal illumination. The following procedure was used for the introduction of the alga into soil. Twenty grams of soil, from which the alga had been isolated initially, were placed in a deep Petri dish and drysterilized at 160 C for 2 hr, and then allowed to cool. Fifty ml of a young, actively growing culture of Spongiochloris typica, with approximately 2 million cells/ml, were added aseptically. Thorough mixing was achieved by swirling the dish. The cover was then propped up slightly to facilitate drying. The dish was incubated under continuous illumination and was visibly dry within 1]0 days.