soil, the heated-soil extract was reduced to almost exactly the same condition as the extract of the same soil before heating.

(b) QUALITATIVE

We next undertook to investigate the nature of the substances that seemed to make heated-soil extracts favorable culture media for Pyronema and other fungi. The pungent odor of the extracts of heated-soil together with the pronounced acidity towards litmus suggested acids, while the dark color and caramel odor suggested carbohydrates or their decomposition products. The following tests were made on heated-soil extracts before being evaporated: Litmus paper-red.

Lead acetate-brownish precipitate.

Silver nitrate-slight precipitate (not soluble in ammonia). Barium chloride-slight precipitate.

Alcohol-slight precipitate.

Calcium hydroxide-slight precipitate.

Ether does not dissolve color of solution.

Molisch test-positive.

This same extract when evaporated to one fiftieth its original bulk showed the same reactions in every case except that they were far more pronounced. This concentrated extract also caused a strong reduction of Fehling solution while the blanks. were negative. All of the above tests were repeated many times and the results were practically always in accord with those described above.

When unheated-soil extracts were tested in exactly the same manner, the acidity was slight as shown by litmus, barium chloride gave a slight precipitate (owing to sulphates), and silver nitrate also gave a slight precipitate wholly soluble in ammonium hydroxide (probably owing to chlorides), while all the other tests were negative.

From the qualitative tests just described we are inclined to believe that upon heating to about 160° to 180° C., the organic matter in the soil undergoes some deep-seated changes probably oxidative in nature, favored by the high temperatures, which give us the water-soluble products of an acid character producing the dark-colored solutions. The acidity of heated-soil extracts

and the heavy precipitates obtained with lead acetate, silver nitrate, and calcium hydroxide, might well be due to the presence of organic acids. The positive Molisch test indicates carbohydrates or their decomposition products, while the strong reducing action on Fehling solution would seem to confirm the assumption that carbohydrate substances are present. It is not at all impossible that the partially disintegrated cellulose of the bodies of plants previously growing on the soil, would be broken up into still smaller fragments of the original enormous molecule, and that these smaller fragments would still retain some of their carbohydrate characteristics together with the added one of acidity.

We next examined the ash of the North Dakota heated-soil extract in a qualitative manner to discover if possible the nature of the inorganic substances in the extract. The ash of the North Dakota soil was used for the reason that this was obtained in considerable quantity and that the extract of this soil was unusually favorable as a culture medium for fungi, probably owing to the large amount of organic matter originally present in the soil. The ash of the unheated North Dakota soil was pure white and soluble in water (100 c.c.). Upon analysis the ash was found to consist principally of the sulphates of sodium, potassium, magnesium and calcium; we were however able to find scarcely a trace of phosphates. About one-half of the ash of the extract of heated North Dakota soil was found to be soluble in water. In the soluble portion of the ash, sulphates of potassium, sodium, magnesium and calcium, etc., were found. In the insoluble part of the ash, we found principally calcium sulphate with some manganese, iron and traces of phosphates, etc. Calcium is thus seen to be present in considerable quantities in the extracts of heated- and unheated-soils and it may be, from the well-known stimulating and protective properties of calcium toward plants, that this element along with the organic matter helps to give heated soils some of their striking properties.

Just as this work was being brought to a close we received, upon request, a copy of an article (in galley proof) by Professor T. L. Lyon, who is publishing the results of his investigations on the effects of steam sterilization upon soils. With steam heat he found the same great increase of soluble matter over

unheated-soils that we found in the case of dry heat at 180° C. He also calls attention to the same disappearance of this soluble organic matter when soils were allowed to stand after steam sterilization, that we had noted in our work on Pyronema. All this shows that either dry or steam heat may cause very important changes in soils and that it is to the effect of these changes on plants, as well as to the destruction of bacteria, etc., that we must ascribe the cultural results often noted in our experiments with heated-soils.

VIII.

PRODUCTS OF DRY DISTILLATION OF SOIL

Steam

In order to see if the heating of a soil would drive off substances toxic to Pyronema, we filled a combustion tube with soil, put it in the furnace, fitted to the tube a smaller glass tube opening under a receiver of distilled water and heated the soil. came over first and then more and more of a yellowish oil which was partially suspended in the water and partially formed a scum on the surface. The oily substance had an intensely irritating and nauseating odor like that of an old, stale pipe and recalled pyridine or its allies. The liquid in the receiver was alkaline to litmus. All of this seemed to indicate pyridine bases. We watered some heated-soil with this liquid and inoculated it with Pyronema. In a week, the growth on this soil was as good as that on the control watered with distilled water.

The soil left in the tube was black. This was watered with distilled water and inoculated with the spores of Pyronema but proved to be unfavorable to its growth, although some mycelium. was produced.

IX. IDENTITY OF THE FUNGUS

The fungus which we have been cultivating in the laboratory had been determined by us as Pyronema omphalodes (Bull.) Fuckel, although its appearance in the laboratory differed slightly from specimens of this species previously observed by us in the field. In nature the ascocarps of this species give rise to dense, confluent masses in which it is difficult to recognize the individual ascocarps, while in the laboratory the plants are thickly gregarious but not confluent to the extent that they are in nature. It therefore occurred to us that the species might be distinct.

PLATE XXVI

MYCOLOGIA

Both pots of soil heated, the one to the left watered with heated-soil extract and the one to the right with distilled water; to show the improvement of heated soil for Pyronema by the addition of more extract.