CHAPTER XLI.

MINERAL NUTRIENT MATERIALS.

§ 228.-Alkalis.

A GLANCE through the existing analytical data concerning the ash constituents of fungi (see for example the handbook by J. KŒNIG (I.)) will soon reveal that the chief of these constituents are phosphoric acid and potash. The latter seldom forms less than one quarter, and is generally about one half of the total weight of ash, sometimes even rather more; the ash of truffles, for instance, according to an analysis by LIEBIG (II.), contains 54.5 per cent. of K2O and 33.0 per cent. of P2O5. From this circumstance alone it may be concluded that, as has already been placed beyond doubt in the case of green plants, potassium is also of importance to fungi. The first experiment made in order to clear up this question was instituted by NÆGELI (IV.), and the results obtained led this worker to assume that potassium is so far non-essential to the growth of fungi that it can be replaced by rubidium or cæsium; but that one or other of these three must invariably be present.

With regard to cæsium, all subsequent investigators, however, agree that this metal is unsuitable for replacing potassium for the purpose in question. Instances of this are furnished by S. WINOGRADSKY (XI.) in his culture experiments with Mycoderma vini; W. BENECKE (II.) in the case of Penicillium glaucum and Aspergillus niger; and subsequently by E. GUENTHER (I.) for Mucor corymbifer, Rhizopus nigricans, and Botrytis cinerea.

Opinions are divided as to the suitability of rubidium for replacing potassium. An affirmative result was furnished by O. LOEW'S (VII.) culture experiments with a species of Penicillium, and by Winogradsky with the film-fungi already mentioned. On the other hand, in the experiments of W. BENECKE (II.) potassium was found replaceable by this allied metal, but only in cases where merely vegetative development was in question. Finally, the experiments of E. GUENTHER (I.) furnished no uniform results: the cultivation in solutions containing rubidium, but no potassium, being successful in the case of Botrytis cinerea, but not so with Rhizopus nigricans. Now, in order to rightly appreciate these results it will be necessary to bear in mind the great difficulty experienced in completely freeing the

rubidium salt from the accompanying potassium salts. This difficulty is, moreover, accentuated by the comparatively ready solubility (even at simple boiling temperature) of the glass of the culture vessels, the consequence being that a little alkali finds its way into the nutrient solution from the glass during the sterilising process. In order to eliminate this source of error several workers have already recommended the use of metallic culture vessels for the purpose in question, but soon had to abandon same on account of the toxic action and consequent retardation of development produced on the sowing. This was observed, in the case of silver, by RAULIN (III.), whose similar observation in the case of tin was confirmed by W. BENECKE (III.). This last-named worker also excluded aluminium from the list of suitable metals, for the reason that it sustained corrosion and therefore caused an alteration of the nutrient solution, although TH. BOKORNY (I.) employed it, apparently with good results, in his experiments on the nutrition of algae. Finally platinum, which, according to Bokorny, has a poisonous effect on the green thallophytes in question, was found by BENECKE to be innocuous in the case of Aspergillus niger; it is, however, too expensive to use for large series of experiments. The difficulties encountered in purifying the nutrient salts, in order that the experiments conducted therewith may be perfectly reliable, can best be appreciated by the aid of the following data, for which we are indebted to W. BENECKE (II.) and E. GUENTHER (I.). The figures relate to the minimum quantity of KCl which will enable the development of the sowing to proceed when added to 100 c.c. of a nutrient solution previously free from potash. Aspergillus niger is sensitive to 0.02 mg., Rhizopus nigricans to 0.01 mg., Mucor corymbifer to 0.02 mg., and Botrytis cinerea to 0.01 mg. of KCl. The essential requirements of the fungi in respect of potash are therefore very moderate; and, in fact, if the necessary quantity be exceeded, to the extent of several units per cent., the growth may be injured. The maximum amount of potash salts which Rhizopus nigricans will stand, and still continue not only to grow but also to fructify, has been determined by E. GUENTHER (I.) as follows: KCl, about 7.5 per cent.; KNO, about 7 per cent.; whilst in the case of K2SO4 the organism will still bear up to 10 per cent. (concentrated solution) very well.

In opposition to the concordant results obtained by earlier workers, CARL WEHMER (IV.) assumed that sodium is able to replace potassium as a nutrient material for fungi. This view was, however, disproved by the experiments of W. BENECKE (III.) on Aspergillus niger, an undescribed species of Penicillium, Mucor stolonifer, Botrytis cinerea, and a pure-culture wine yeast from Winningen; these results being also strengthened by E. GUENTHER'S (I.) culture experiments with Mucor corymbifer,

The rule therefore

Rhizopus nigricans, and Botrytis cinerea. still holds good that sodium is of no appreciable utility as regards the nutrition of fungi, and can be entirely dispensed with. The maximum quantities of the salts of this metal that can be present in the nutrient solution without injury, have been determined by E. Guenther, in the case of Rhizopus nigricans, as follows: NaCl, 12 per cent.; Na2SO4 + 10 aq., 26 per cent.; and NaNO3, 6 per cent.

With regard to lithium, W. BENECKE (III.) has shown-in refutation of the contrary assumption by Naegeli-that this metal is not a foodstuff for fungi, although a strong stimulant. When lithium salts were present in the nutrient solution it was found that the conidia of Aspergillus niger did not germinate, and that no conidia were formed in the case of an unspecified species of Penicillium. The extent to which the various species are sensitive to the action of this metal must fluctuate considerably, since, whilst E. GUENTHER (I.) found 0.05 per cent. to be the largest addition of lithium nitrate that Rhizopus nigricans could stand and still continue to thrive, H. M. RICHARDS (I.) was able to observe that Aspergillus niger gave a crop of double the usual size when the nutrient solution of saccharose and mineral salts was treated with an addition of 0.3 to 0.5 per cent. of lithium chloride.

$ 229. Metals of the Alkaline Earths.

According to NÆGELI (IV.) magnesium is non-essential for the development of fungi when the latter have at disposal one of the three alkaline earths: strontium, barium, or calcium. The assumption that fungi can grow without magnesium has, however, been disproved by WINOGRADSKY (XI.), who showed that the latter is indispensable for the development of Mycoderma vini. The same results were obtained by Adolf Mayer in culture experiments with beer yeast; by H: MOLISCH (II.) and W. BENECKE (I.) with Penicillium glaucum and Aspergillus niger ; and by E. GUENTHER (I.) with Mucor corymbifer, Rhizopus nigricans, and Botrytis cinerea. How sensitive and responsive the fungi are to a small addition of magnesium is evident from the observation recorded by BENECKE (III.) as to the considerable difference in development exhibited by two, otherwise equal, specimens, the one grown without magnesium and the other in a medium containing 0.0025 mg. of crystalline magnesium sulphate per 25 c.c. Similarly, E. GUENTHER (I.) ascertained, in the case of magnesium sulphate, (MgSO4+7 aq.), that a minimum addition of 0.005 mg. was necessary to induce a sowing of Rhizopus nigricans to grow at all.

In refutation of an earlier assumption by Sestini, it has been shown by H. MOLISCH (II.) and W. BENECKE (I.), and after

wards confirmed by E. GUENTHER (I.), that not only calcium, barium, and strontium, but also the closely allied metals beryllium, zinc, and cadmium are unsuitable for replacing magnesium; and that, in fact, they behave as poisons when added in slightly larger amount to the nutrient medium. An addition of 0.02 per mil of cadmium sulphate or cadmium chloride is sufficient for Aspergillus and Penicillium; whilst for Rhizopus nigricans 0.001 per mil is enough. According to Guenther, an addition of 0.2 per cent. of beryllium chloride is necessary to restrict the development of the last-named Phycomyces.

With regard to zinc, it was observed by J. RAULIN (I. and III.) before 1870, in his experiments with Aspergillus niger, that the mycelial development of this fungus could be considerably facilitated by a small addition of zinc sulphate to the nutrient medium. The conclusion drawn therefrom that, in contradistinction to the earlier discoveries of the same worker, zinc is indispensable to the structure of the fungus in question was, however, unable to stand subsequent investigation. Both in this fungus and in the case of Penicillium glaucum and Botrytis cinerea it was found by W. PFEFFER (II.), H. M. RICHARDS (I.), and ONO (I.), that the action of zinc is stimulative (§ 81) in the sense of H. SCHULZ's law (I.). Even an addition of 0.0005 per cent. of zinc sulphate to a nutrient solution of, e.g. saccharose and mineral salts, resulted in a considerable increase in cropping. This attained double the yield (furnished in the absence of zinc) when the addition reached the optimum amount of about 0.003 per cent. of zinc sulphate; but, on raising the addition to 0.05 per cent., a poisonous action was observed. Rhizopus nigricans seems to be still more sensitive, since, according to E. GUENTHER (I.), it will not stand more than 0.01 gram of zinc sulphate in 100 c.c. of nutrient solution. A noteworthy observation made by several workers is that this stimulation is really a kind of fattening process, the stimulative influence being confined to the development of the mycelium, that is, to the vegetative portion of the thallus; whilst the production of conidia, or organs of fructification, is retarded, and even entirely suppressed. Supported by other experimental results (relating to copper as well as zinc), ANDR. RICHTER (I.) has pointed out that, in such a state of dilution, the salts (e.g. zinc sulphate) are no longer capable of acting as such, but-in accordance with the theory of dissociation— are more or less separated into their components, the ions Zn and SO, which are therefore the real stimulants. Moreover, because the extent of the dissociation is also dependent upon the nature of the solvent-in this case the nutrient solutionthe action exerted by an addition of such saline stimulants is also determined thereby. The present is a suitable occasion for referring to the labours of TH. PAUL and B. KRŒNIG (I. and II.), SCHEURLEN and SPIRO (I.), and others, to whom we are indebted

-since the publication of vol. i.-for the application of the dissociation theory to the study of the action of poisons on microorganisms (§§ 79 and 81), and for the useful conclusions that may be drawn therefrom with regard to practical disinfection.

In connection with magnesium, indirect reference has already been made to calcium, inasmuch as Naegeli's assumption that this metal could replace magnesium was stated to have been refuted. There still remains, however, the less important question whether calcium is at all essential to the structure of fungi. On this point Winogradsky, in a previously cited research, found Mycoderma vini still capable of thriving in a nutrient medium free from calcium (and also from barium and strontium). The same results were obtained by H. MOLISCH (II.) and W. BENECKE (II.) in their experiments with Aspergillus niger so we may well assume that calcium is not essential to the growth of fungi. Until a short time ago it was considered that this peculiarity afforded a thorough means of distinction between the fungi and green plants. However, it has recently been found by H. MOLISCH (IV.) that certain (but not all!) of the algae will thrive in the absence of calcium, e.g. Stichococcus bacillaris Nægeli, Ulothrix subtilis Kuetzing, but not Vaucheria or Spirogyra. For the future, therefore, the axiom must be changed, and calcium regarded as indispensable for the higher green plants, but not essential to the fungi and to certain algæ.

So far as barium and strontium are concerned, it has been placed beyond doubt that these two metals are not only useless, but also injurious, and act as poisons toward the fungi. Thus, for example, in the experiments of E. Guenther, the development of sowings of Rhizopus nigricans ceased in presence of 1.0 per cent. of barium nitrate, or 1.5 per cent. of strontium nitrate, in the nutrient solution. Even calcium, it may be remarked in passing, is capable of acting injuriously when present in larger quantities, the last named worker having found the limit of safety to be 4 per cent. of calcium nitrate in the case of the same Phycomyces.

§ 230.-Elements of the Iron Group.

On the fact that iron had been shown indispensable to the formation of chlorophyll, and therefore essential to the assimilative power of green plants, was based the opinion, long unanimously current among mycologists-e.g. ADOLF MAYER (I.) in connection with yeast, and C. Nægeli-that the fungi, being destitute of chlorophyll, do not require iron. This assumption was first put to the test by J. RAULIN (I.) in 1870, the results showing that, in presence of iron, cultures of Aspergillus niger throve more vigorously in solutions of nutrient salts containing