THE FUNGOUS FLORA OF PINE SEED BEDS ANNIE E. RATHBUN PART I. FUNGOUS FLORA OF THE SOIL I. Introduction About four years ago a nursery for seedlings of Pinus resinosa Ait. and P. ponderosa Dougl. was established at the Metcalf Botanical Garden of Brown University, Providence, R. I. Each season almost all of the seedling pines have damped off. The writer began the present problem, a preliminary account of which is given in this paper, with the hope of ascertaining whether the soil of the nursery contains any fungi, besides Fusarium, which are capable of producing damping off. An attempt was also made to find out at what depth. the various fungi occur and how widely they are distributed in the nursery and the surrounding meadow. Later, for the sake of comparison, a brief study was made of two lots of soil from another part of the state. In recent years many fungi have been isolated from the soil, but very few, so far as can be learned, have been reported definitely below a depth of 8 inches. Waksman (1) states that he found Zygorrhynchus vuillemini at all depths from 1 to 30 inches, but that most of the "other organisms" were obtained from the upper 8 inches. He adds "in most samples taken at depths of 12, 20 or 30 inches only Zygorrhynchus would develop from the soil upon the plate, with no other organism." Miss Taylor (2) has reported Fusarium at practically every depth from 1 to 24 inches. Most other investigators of soil fungi either procured their samples from the upper 8 inches of soil or do not state at what depth they took them. Goddard (3) discovered Fusarium, Mucor, and Trichoderma at various depths to 14 cm., but does not mention at what depths he found the other fungi which he describes. Werkenthin (4) collected soil from 1 to 7 inches, but states that he found no fungous organisms at depths of 5, 6 and 7 inches. He adds that the same species of fungi are found from each of the first 4 inches. Jensen (5) also made isolations from the upper 8 inches of soil, but does not report at what depths the various fungi occurred. Beckwith (6) obtained the material for his investigations 2 inches below the surface. McLean (7) (8) and Miss Dale (9) do not mention the particular depths at which they found fungi. II. Present investigations A. Description of soil from which samples were collected. 1. Soil at the Metcalf Botanical Garden, Providence, R. I. The land is almost 'evel, sloping gently to the east and the northeast. The top soil consists of a layer of medium light colored, sandy loam, which is from 10 to 14 inches in depth. Beneath this is a bed of coarse yellow sand from 3 to 10 feet deep. This is underlaid by hardpan. 2. Soil of the R. H. I. Goddard Estate, East Greenwich, R. I. This estate is situated about 15 miles in a straight line from the Botanical Garden. The top soil of that portion of the estate where lot F was collected is rich, dark, sandy loam about 2 feet in thickness. Lot G was procured from top soi' consisting of light sandy loam, running to a depth of 8 to 10 inches. Both top soils are underlaid with sand and gravel to a depth of at least 10 feet. B. Methods and time of obtaining soil samples. Several lots of soil, A, B, C, D, E, F and G were collected, each sample of each lot containing to 1 pint of soil. The first (A) was taken in October and the second (B) in December, 1916; the third (C) in January, the fourth (D) in February and the fifth (E), the sixth (F) and the seventh (G) in March, 1917. Lots A, B, C, D and E came from the nursery while lots F and G were taken from the R. H. I. Goddard Estate, East Greenwich, R. I. Lot A was collected in meadow land which has not been cultivated for a period of twenty years. It was ploughed in the spring of 1916 and has not been cultivated since. This meadow land is situated near the fence on the south side of the nursery about 75 feet from the pine seed bed where all the seedlings had damped off. Lot C came from sod land which was ploughed in the spring of 1913 and has since remained uncultivated. When this collection was made the soil was frozen to a depth of 10 inches. Lot D was procured from the meadow land along the fence at the northeast corner of the nursery about 75 feet from the pine seed beds. The upper 14 inches of this soil were frozen. Lot E was collected in the meadow land from which lot A came, about 15 feet southeast of the entrance to the nursery and about 10 feet from pine seed beds in which damping off had occurred. Lot F came from a vegetable garden at East Greenwich and Lot G from a nearby garden which was planted with corn in 1916. The soil of both gardens had been heavily fertilized with stable dressing almost every year, while peat muck had also been added to the latter. The ground had thawed to a depth of 6 to 8 inches at the time the samples were taken. The soil samples were collected in the following manner. First a trench, the dimensions of which were about 4 feet by 2 feet by 5 feet was dug. The trench was always 5 or 6 inches deeper than the lowest depth at which soil was collected. One side of it was dug perpendicular. A series of undercuts about 3 inches apart were made beginning at the bottom of the trench. The soil was scraped by means of a sterile scraper from the under side of the shelf and allowed to drop into sterilized baking powder cans or Mason jars. Thus the danger of contamination from above was greatly minimized. C. Media. Five kinds of agar-soil extract, oat meal, corn meal, prune and pine twig-were used. 1. The soil agar was prepared according to the formula described by Jensen (5) as "soil extract agar II." This medium was most satisfactory for both poured plates and stab cultures. Upon it all the fungi produced characteristic colonies, while Fusarium usually caused it to turn very rosy red. The soil extract gave the best results when freshly prepared. 2. The oat meal agar was prepared according to a formula in general use in the botanical laboratory of Brown University. The fungi grew upon this medium almost as well as on soil agar, but the color reaction in the case of Fusarium was less marked. Because of its opaqueness, it was less convenient for poured plates. 3. Corn meal agar was made by substituting Rhode Island corn meal — a white unbolted meal-for the oat meal of the above medium (2). This agar was very unsatisfactory for poured plates and, even in case of stab cultures, little additional data could be secured by its use. 4. Pine twig agar was prepared according to Shear's (10) formula for chestnut twig agar except for the fact that shredded agar melted over a free flame was used in place of agar flour melted by steaming. This agar was tried out primarily to ascertain whether any of the soil organisms would grow especially well on a medium containing an extract of pine twigs. Fusarium, Zygorrhynchus, and some of the other fungi fruited upon it in tubes. Upon poured plates the colonies were small and of little variety. It was not satisfactory as a whole. 5. Prune agar was made according to Shear's (10) prune agar formula with the modification noted above for pine twig agar. This agar was very unsatisfactory, for few colonies grew upon it. Bacteria were more troublesome on this medium than on the others. D. Isolation from the soil. The cultures were made in the manner described below. A can of soil was taken into the culture chamber and its contents was emptied into a sterilized Mason pint jar and shaken vigorously. With a sterilized spatula, which held approximately 0.55 of a gram, varying slightly with amount of moisture present, soil was taken out and placed in a bottle containing 100 cc. of distilled water. Each bottle was shaken well and, before the soil had time to settle, 1 cc. of the mixture was taken out by means of a sterile pipette. This was added to 10 cc. of melted soil agar and then plated. At least two plates were made for each sample of each lot. In order to test the growth of the fungi on other media and to ascertain whether other organisms could be secured, additional plates were made from time to time. In case of lot D, not only additional media, but also an additional method of plating was used. One cubic centimeter of the soil solution was poured into a Petri dish. To this was added 10 cc. of melted soil agar and the two were mixed by a rotatory movement of the Petri dish. Since no more colonies were secured by this method than by the former, it was later abandoned. Fungous and bacterial colonies usually began to appear at the end of two days. At this time the plates were examined and the fungi isolated by the ordinary bacterial method for obtaining pure cultures. The bacterial colonies were very abundant at first, but within four or five days the fungous colonies had overgrown them. III. Results of the present investigation A variety of fungi appeared in all the plates. It is assumed that all those forms appearing are soil forms, but contamination may have taken place in some instances, in spite of all care taken to avoid it. Some of the slow growing fungi were crowded out by the rapid growing forms; some were undoubtedly lost in the process of isolation, while others still await identification. The following tables give a list of the fungi which have been identified thus far from the various depths and, except in the case of lot A, the approximate number of colonies which appeared on the various plates. An asterisk (*) in the column following the name of a fungus indicates the depth at which it was found and shows that no estimate of colonies was made. |