Nitrogen Relationships.
In this section we shall consider the problems of nitrogen fixation and nitrification, of ammonification, and of the utilisation of nitrogenous compounds by soil fungi.
As soil fungi form so large a part of the soil population, the question of whether they can make use of the free nitrogen of the air is of primary importance. During the last two decades many investigators have attempted to solve the problem, often studying allied or identical species; but if one consults some thirty researches published during this period, opinion is found to be about equally divided. Even, however, in those studies where nitrogen fixation has been recorded the amounts are very slight, usually being below 5 mgrms. per 50 c.c. of solution, and often being obviously within the limits of experimental error. Latham,[37] however, working on Aspergillus niger, recorded variations ranging from a nitrogen loss of 42·5 mgrms. to a nitrogen fixation of 205·1 mgrms. per 50 c.c. of medium. Ternetz[63] found that different strains of Phoma radicis may fix from 2·5 mgrms. of nitrogen in the lowest case, to 15·7 mgrms. in the highest per 50 c.c. of nutrient solution. Duggar and Davis[20] report that Phoma betæ may fix nitrogen in quantities of 7·75 mgrms. per 50 c.c. of medium. The latter authors, in a very able critique of the problem, indicate certain possible sources of error in previous work, and if one examines the studies in which nitrogen fixation has been recorded in the light of these criticisms, it is difficult not to think that, with the exception of the genus Phoma, good evidence for nitrogen fixation by fungi is lacking. Phoma betæ is a common pathogen attacking beets, whilst P. radicis is a mycorrhizal form inhabiting various Ericales. Apart from these exact quantitative studies, which have given a negative verdict, there is a considerable amount of positive but indirect evidence for nitrogen fixation by mycorrhizal fungi,[55] and it is very unfortunate that more of these forms have not been investigated quantitatively. As the evidence stands to-day, one must conclude that the fungus flora does not play any part in the direct nitrogen enrichment of the soil.
Equally obscure is the question of nitrification and denitrification by soil fungi, but this is the result of a lack of study rather than of a plethora of indeterminate researches. Direct nitrification or denitrification has not been established, but the work of Laurent[38] and a few other workers appears to show that soil fungi can reduce nitrates to nitrites.
The second primary nitrogen relationship that we have to consider is the process of ammonification. The ammonifying power of soil fungi was first demonstrated by Muntz and Coudon,[46] and by Marchal[40] in 1893, the former showing that Mucor racemosus and Fusarium Muntzii gave a larger accumulation of ammonia in soil than any of the bacteria tested; and the latter that Aspergillus terricola, Cephalothecium roseum and other soil fungi were active ammonifiers, especially in acid soils. Shibata,[62] Perotti,[49] Hagem,[26] Kappen,[31] Löhnis,[39] and others, have observed that urea, dicyanamide and cyanamide are decomposed with the liberation of ammonia; and Hagem[26] has recorded the same process for peptones, amino acids, and other organic nitrogen compounds in plant and animal remains in the soil. The latter author considers soil fungi more important ammonifying agents in the soil than bacteria, a conclusion in which McLean and Wilson,[44] and perhaps most later workers concur. McLean and Wilson[44] found large differences in the ammonifying powers of various soil fungi, the Moniliaceæ being the strongest ammonifiers, the Aspergillaceæ the weakest. Generic and specific differences have been confirmed by Coleman,[15] Waksman,[67] and other authors. Waksman and Cook[70] suggested that such variations may be due, not to innate differences in the metabolic activities of the several organisms, but to differences in reproductive times, and that there might be some relationship between sporogeny and the ability to accumulate nitrogen. Kopeloff[35] has carried out experiments on the inoculation of sterilised soil with known quantities of spores and found that, although the amount of ammonia accumulated increased with the number of spores the proportion was not direct but modified by the food supply. After the first five days’ growth, the rate of ammonia production varied markedly in a two-day rhythm which seemed to be due to the metabolism of the fungus rather than to recurrent stages of spore formation and germination in the life history. The amount of ammonia liberated has been shown by recent work[66] to depend upon the available sources of carbon and nitrogen. In the absence of a carbohydrate supply the protein is attacked both for carbon and nitrogen, and since more of the former is required much ammonia is liberated. In addition, however, to the carbon and nitrogen control, the process of ammonification by soil fungi is intimately related to physical conditions. Working with pure cultures, McLean and Wilson,[44] Coleman,[15] Kopeloff,[35] Waksman and Cook,[70] and other students, have shown that the amount of ammonia accumulated depends upon such factors as the presence of phosphates, the period of incubation of the fungi, aeration, the moisture in the soil, the temperature, the degree of soil acidity, the type of soil, and so forth.
That fungi take a very important place as ammonifying agents in the soil can no longer be doubted, but the question yet remains to be considered of the balance of profit or loss resulting from their activities. It has usually been considered that a part of the ammonia freed is used by the fungi themselves, but that the greater part is liberated, and so rendered available to nitrifying organisms. Both Neller[47] and Potter and Snyder[51] found that typical soil fungi inoculated into sterile soil grew with a vigour approximately equal to the growth induced by an inoculation of the entire soil flora. This is largely to be accounted for by the fact that when soils are sterilised by heat or by certain chemicals, breaking-down changes occur, and substances are liberated which are peculiarly favourable to fungus growth. This fact must be borne in mind when interpreting ammonification and other studies where the method is that of inoculation of fungi into sterilised soil. In many cases it tends to nullify any application of the results to normal soils, whilst in others the conclusions must be accepted with some reserve. In all cases Potter and Snyder[51] found that fungi caused a diminution in the amount of nitrates, that the ammonia was not much changed in amount, and that there was a decrease in the quantities of soluble non-protein nitrogen. The range of organic and inorganic nitrogenous compounds utilisable by soil fungi is very great. Ritter[56] has shown that certain forms can use the nitrogen of “free” nitric acid in the medium; Ritter,[56] Hagem,[26] and others, that soil fungi can use ammonia nitrogen equally with nitrate nitrogen, and Ehrenberg[21] concluded that soil fungi play a more important part in the building of albuminoids from ammonia than bacteria do. Ehrlich[22] has shown that various heterocyclic nitrogen compounds and alkaloids can serve as sources of nitrogen to soil fungi, whilst Ehrlich and Jacobsen[23] have found that soil fungi can form oxy-acids from amino-acids. Hagem,[26] Povah,[52] Bokorny,[6], [8] and others, state that for many soil forms organic nitrogen sources are better than inorganic sources, and that peptones, amino-acids, urea, and uric acids, etc., are very quickly utilised by species of Mucor, yeasts, and so forth. Butkevitch,[12] and Dox[18] have recently found that it depends on circumstances which compounds of protein molecule can be utilised by particular fungi, and that soil fungi can utilise both amino and amido complexes for the formation of ammonia. In 1919 Boas[4] showed for Aspergillus niger that if a number of nitrogenous compounds are available the fungus absorbs the most highly dissociated.
In the welter of scattered observations on the utilisation of nitrogenous compounds, it is difficult to trace any clear issue. That proteins, amino-acids, and other complex organic compounds are readily broken down to ammonia by soil fungi is clear, and, on the other hand, it is also clear that soil fungi utilise extensively ammonia and nitrates as sources of nitrogen. On which side the balance lies it is yet impossible to say.