Part 18

The germination of the pseudospores of the gelatinous Uredines of the genus Podisoma was studied by Tulasne. These pretended spores, he writes, are formed of two large conical cells, opposed by their base and easily separating. They vary in length. The membrane of which they are formed is thin and completely colourless in most of them, though much thicker and coloured brown in others. It is principally the spores with thin membranes that emit from near the middle very obtuse tubes, into which by degrees, as they elongate, the contents of the parent utricles pass. Each of the two cells of the supposed spore may originate near its base four of these tubes, opposed to each other at their point of origin, and their subsequent direction; but it is rather rare for eight tubes, two by two, to decussate from the same spore or basidium. Usually there are only two or three which are completely developed, and these tend together towards the surface of the fungus, which they pass, and expand at liberty in the air. The tubes generally become thicker by degrees as they elongate, some only slightly exceeding the length of the protospores. Others attain three or four times that length, according to the greater or less distance between the protospore and the surface of the plant. In the longest tubes it is easy to observe how the colouring matter passes to their outer extremity, leaving the portion nearest to the parent cell colourless and lifeless. When nearly attaining their ultimate dimensions, all the tubes are divided towards their outer extremity by transverse septa into unequal cells; then simple and solitary processes, of variable length and form, but attenuated upwards, proceed from each segment of the initial tube, and produce at their extremity an oval spore (teleutospore, Tul.), which is slightly curved and unilocular. These spores absorb all the orange endochrome from the original tubes. They appear in immense numbers on the surface of the fungus, and when detached from their spicules fall upon the ground or on any object which may be beneath them. So freely are they deposited that they may be collected on paper, or a slip of glass, like a fine gold-coloured powder. Again, these secondary spores (teleutospores) are capable of germination, and many of them will be found to have germinated on the surface of the Podisoma whence they originated. The germ filament which they produce springs habitually from the side, at a short distance from the hilum, which indicates the point of attachment to the original spicule. These filaments will attain to from fifteen to twenty times the diameter of the spore in length before branching, and are in themselves exceedingly delicate. The tubes which issue from the primary spores (protospores, Tul.) are not always simple, but sometimes forked; and the cells which are ultimately formed at their extremities, though producing filiform processes, do not always generate secondary spores (teleutospores) at their apices. This mode of germination, it will be seen, resembles greatly that which takes place in Puccinia.

The germination of the Ustilagines was in part examined by Tulasne, but since has received accessions through the labours of Dr. A. Fischer von Waldheim. Nothing, however, of any importance is added to our knowledge of the germination of Tilletia, which was made known as early as 1847. After some days a little obtuse tube is protruded through the epispore, bearing at its apex long fusiform bodies, which are the sporules of the first generation. These conjugate by means of short transverse tubes, after the manner of the threads of Zygnema. Afterwards long elliptical sporules of the second generation are produced on short pedicels by the conjugated fusiform bodies of the first generation. (Fig. 89, ss.) Ultimately these sporules of the second generation germinate, and generate, on short spicules, similar sporules of a third generation. (Fig. 89, st.)

In Ustilago (flosculorum) germination takes place readily in warm weather. The germ tube is rather smaller at its base than further on. In from fifteen to eighteen hours the contents become coarsely granular; at the same time little projections appear on the tube which are narrowed at the base, into which some of the protoplasm passes. These ultimately mature into sporules. At the same time a terminal sporule generally appears on the threads. Secondary sporules frequently grow from the primary, which are rather smaller, and these occasionally give rise to a third generation.

In Urocystis (pompholygodes) the germinating tubes spring exclusively from the darker central cells of the clusters. From these are developed at their extremity three or four linear bodies, as in Tilletia, but after this no further development has as yet been traced. It may be remarked here that Waldheim observed similar conjugation of the sporules in some species of Ustilago as have been remarked in the sporules of the first generation in Tilletia.

Returning to Cystopus, as the last of the Uredines, we must briefly recapitulate the observations made by Professor de Bary, who, by the bye, claims for them an affinity with Peronospora (Mucedines but too well known in connection with the potato disease), and not with the Uredines and their allies. In this genus there are two kinds of reproductive organs, those produced on the surface of the plant bursting through the cuticle in white pustules, and which De Bary terms conidia, which are generated in chains, and certain globose bodies termed oogonia, which are developed on the mycelium in the internal tissues of the foster plant. When the conidia are sown on water they rapidly absorb the moisture, and swell; the centre of one of the extremities soon becomes a large obtuse papilla resembling the neck of a bottle. This is filled with a granular protoplasm, in which vacuoles are formed. Soon, however, these vacuoles disappear, and very fine lines of demarcation separate the protoplasm into from five to eight polyhedric portions, each presenting a little faintly-coloured vacuole in the centre (a). Soon after this division the papilla at the extremity swells, opens itself, and at the same time the five to eight bodies which had formed in the interior are expelled one by one (b). These are zoospores, which at first take a lenticular form, and group themselves before the mouth of the parent cell in a globose mass (c.) Very soon, however, they begin to move, and then vibratile cilia show themselves (d), and by means of these appendages the entire globule moves in an oscillating manner as one by one the zoospores disengage themselves, each becoming isolated and swimming freely in the surrounding fluid. The movement is precisely that of the zoospores of Algæ.

The generation of the zoospores commences within from an hour and a half to three hours after the sowing of the conidia on water. From the oogonia, or resting spores, similar zoospores, but in greater number, are generated in the same manner, and their conduct after becoming free is identical. Their movements in the water usually last from two to three hours, then they abate, the cilia disappear, and the spore becomes immovable, takes a globose form, and covers itself with a membrane of cellulose. Afterwards the spore emits, from any point whatever of its surface, a thin, straight or flexuous tube, which attains a length of from two to ten times the diameter of the spore. The extremity becomes clavate or swollen, after the manner of a vesicle, which receives by degrees the whole of the protoplasm.

De Bary then proceeds to describe experiments which he had performed by watering growing plants with these zoospores, the result being that the germinating tubes did not penetrate the epidermis, but entered by the stomates, and there put forth an abundant mycelium which traversed the intercellular passages. Altogether the germination of these conidia or zoospores offers so many differences from the ordinary germination of the Uredines, and is so like that which prevails in Peronospora, in addition to the fact of both genera producing winter spores or oogonia, that we cannot feel surprised that the learned mycologist who made these observations should claim for Cystopus an affinity with Peronospora rather than with the plants so long associated with it amongst the Coniomycetes.

In passing from these to the Mucedines, therefore, we cannot do so more naturally than by means of that genus of white moulds to which we have just alluded. The erect branched threads bear at the tip of their branchlets spores, or conidia, which conduct themselves in a like manner to the organs so named in Cystopus, and oogonia or resting spores developed on the mycelium within the tissues of the foster plant also give origin to similar zoospores.

The conidia are borne upon erect, elongated filaments, originating from the creeping mycelium. These threads are hollow, and rarely septate; the upper portion divided into numerous branches, and these again are subdivided, the ultimate ramuli each terminated by a single conidium. This body when mature is oval or elliptical, filled with protoplasm, but there is a diversity in their mode of germination. In the greater part, of which P. effusa may be taken as an example, the conidia have the function of simple spores. Placed in favourable conditions, each of them puts forth a germ-tube, the formation of which does not differ in any essential point from what is known of the spores of the greater part of fungi.

The short oval conidia of P. gangliformis have little obtuse papillæ at their apex, and it is at this point that germination commences.

The conidia of P. densa are similar, but the germination is different. When placed in a drop of water, under favourable circumstances, the following changes may be observed in from four to six hours. The protoplasm, at first uniformly distributed in all the conidia, appears strewn with semi-lenticular, and nearly equidistant vacuoles, of which the plane face is immediately in contact with the periphery of the protoplasm. These vacuoles number from sixteen to eighteen in P. macrocarpa, but are less numerous in P. densa. A short time after the appearance of the vacuoles the entire conidium extends itself so that the papilla disappears. Suddenly it reappears, elongates itself, its attenuated membrane vanishes, and the protoplasm is expelled by the narrow opening that remains in place of the papilla. In normal cases the protoplasm remains united in a single mass that shows a clear but very delicate outline. When it has reached the front of the opening in the conidium, which is thus emptied, the mass remains immovable. In P. densa it is at first of a very irregular form, but assumes by degrees a regular globose shape. This is deprived of a distinct membrane, the vacuoles that disappeared in the expulsion again become visible, but soon disappear for a second time. The globule becomes surrounded with a membrane of cellulose, and soon puts out from the point opposite to the opening of the conidium a thick tube which grows in the same manner as the germ-tube of the conidia in other species. Sometimes the expulsion of the protoplasm is not completely accomplished; a portion of it remaining in the membrane of the conidium detaches itself from the expelled portion, and while this is undergoing changes takes the form of a vesicle, which is destroyed with the membrane. It is very rare that the protoplasm is not evacuated, and that the conidia give out terminal or lateral tubes in the manner that is normal to other species without papillæ. The germination just described does not take place unless the conidia are entirely surrounded by water; it is not sufficient that they repose upon its surface. Besides, there is another condition which, without being indispensable, has a sensible influence on the germination of P. macrocarpa, and that is the exclusion of light. To ascertain if the light or the darkness had any influence, two equal sowings were placed side by side, the one under a clear glass bell, the other under a blackened glass bell. Repeated many times, these experiments always gave the same result--germination in from four to six hours in the conidia under the blackened glass; no change in those under the clear glass up to the evening. In the morning germination was completed.

The conidia of P. umbelliferarum and P. infestans show an analogous structure. These bodies, if their development be normal, become zoosporangia. When they are sown upon water, one sees at the end of some hours the protoplasm divided by very fine lines, and each of the parts furnished with a small central vacuole. Then the papilla of the conidium disappears. In its place appears a rounded opening, by which the parts of the protoplasm are expelled rapidly, one after the other. Each of these, when free, immediately takes the form of a perfect zoospore, and commences to agitate itself. In a few moments the sporangium is empty and the spores disappear from the field of the microscope.

The zoospores are oval or semi-oval, and in P. infestans the two cilia spring from the same point on the inferior border of the vacuole. Their number in a sporangium are from six to sixteen in P. infestans, and from six to fourteen in P. umbelliferarum. The movement of the zoospores ceases at the end of from fifteen to thirty minutes. They become motionless, cover themselves with a membrane of cellulose, and push out slender bent germ-tubes which are rarely branched. It is but seldom that two tubes proceed from the same spore. The same development of the zoospores in P. infestans is favoured by the exclusion of the light. Placed in a position moderately lighted or protected by a blackened bell, the conidia very readily produced zoospores.

A second form of germination of the conidia in P. infestans, when sown upon a humid body or on the surface of a drop of water, consists in the conidium emitting from its summit a simple tube, the extremity of which swells itself into the form of an oval vesicle, drawing to itself, little by little, all the protoplasm contained in the conidium. Then it isolates itself from the germ-tube by a septum, and takes all the essential characteristics of the parent conidium. This secondary conidium can sometimes engender a third cellule by a similar process. These secondary and tertiary productions have equally the character of sporangia. When they are plunged into water, the ordinary production of zoospores takes place.

Lastly, there is a third mode of germination which the conidia of P. infestans manifest, and which consists in the conidium emitting from its summit a simple or branched germ-tube. This grows in a similar manner to the conidia first named as of such species as P. effusa. The conditions which control this form of germination cannot be indicated, since some conidia which germinate after this manner will sometimes be found mixed with others, the majority of which furnish zoospores. It may be that the conidia themselves are in some sort of abnormal condition.

In all the species examined the conidia possess the power of germination from the moment of their maturity. The younger they are the more freely they germinate. They can retain this power for some days or weeks, provided they are not entirely dried. Dessication in an ordinary temperature seemed sufficient to destroy the faculty of germinating in twenty-four hours, when the conidia had been removed from the leaves on which they were produced. They none of them retained the faculty during a few months, hence they cannot preserve it during the winter.

The germs of Peronospora enter the foster plant if the spores are sown upon a part suitable for the development of the parasite. It is easy to convince one's self that the mycelium, springing from the penetrating germs, soon takes all the characters that are found in the adult state. Besides, when cultivated for some time, conidiiphorous branches can be seen growing, identical with those to which it owes its origin. Such cultivation is so readily accomplished that it can be made upon cut leaves preserved fresh in a moist atmosphere.

In the species of Peronospora that inhabit perennial plants, or annual plants that last through the winter, the mycelium hidden in the tissues of the foster-plant lasts with it. In the spring it recommences vegetation, and emits its branches into the newly-formed organs of its host, there to fructify. The Peronospora of the potato is thus perennial by means of its mycelium contained in the browned tissue of the diseased tubers. When in the spring a diseased potato begins to grow, the mycelium rises in the stalk, and soon betrays itself by blackish spots. The parasites can fructify abundantly on these little stalks, and in consequence propagate themselves in the new season by the conidia coming from the vivacious mycelium.

The diseased tubers of the potato always contain the mycelium of P. infestans, which never fructifies there as long as the skin of the tuber is intact. But when, in cutting the tuber, the parenchyma occupied by the mycelium is exposed to the contact of the air, it covers itself with conidia-bearing branches at the end of from twenty-four to forty-eight hours. Analogous results are obtained with the stalks of the potato. It is evident that in these experiments nothing is changed except the contact of the air; the specific conditions particularly remain the same. It appears, therefore, that it is this contact alone which determines generally the production of the conidiiferous branches.

The mode of germination and development in the Mucors has been studied by several observers, but most recently by Van Tieghem and Le Monnier. In one of the common forms, the Mucor phycomyces of some authors, and the Phycomyces nitens of others, the process is given in detail. In this species germination will not take place in ordinary water, but it readily takes place in orange juice and other media. The spore loses colour, swells, and absorbs fluid around it until double its original size and ovoid. Then a thick thread is emitted from one or both extremities, which elongates and becomes branched in a pinnate manner. Sometimes the exospore is ruptured and detached loosely from the germinating spore. After about forty-eight hours from the first sowing, the mycelium will send branches into the air, which again become abundantly branched; other short submerged branches will also remain simple, or have tuft-like ramifications, each terminating in a point, so as to bristle with spiny hairs. In two or three days abruptly swollen branches, of a club shape, will make their appearance on the threads both in the air and in the fluid. Sometimes these branches are prolonged into an equal number of sporangia-bearing threads, but most frequently they divide first at their swollen summits into numerous branches, of which usually one, sometimes two or three, develop into sporangia-bearing threads, while the rest are short, pointed, and form a tuft of rootlets. Sometimes these rootlets reduce themselves to one or more rounded protuberances towards the base of the sporangia-bearing threads.

There are often also a certain number of the branches which had acquired a clavate shape, and do not erect themselves above the surface, instead of producing a fertile thread, which would seem to have been their first intention, become abruptly attenuated, and are merely prolonged into a mycelial filament. Although in other species chlamydospores are formed in such places on the mycelium, nothing of the kind has been traced in this species, more than here indicated. Occasionally, when germination is arrested prematurely, certain portions of the hyphæ, in which the protoplasm maintains its vitality, become partitioned off. This may be interpreted as a tendency towards the formation of chlamydospores, but there is no condensation of protoplasm, or investiture with a special membrane. Later on this isolated protoplasm is gradually altered, separating into somewhat regular ovoid or fusiform granules, which have, to a certain extent, the appearance of spores in an ascus, but they seem to be incapable of germination.

Another method of reproduction, not uncommon in Mucorini, is described by Van Tieghem in this species. Conjugating threads on the substratum by degrees elaborate zygospores, but these, contrary to the mode in other species, are surrounded by curious branched processes which emanate from the arcuate cells on either side of the newly-developed zygospore. This system of reproduction is again noticed more in detail in the chapter on polymorphism.

M. de Seynes has given the details of his examination of the sporidia of Morchella esculenta during germination. A number of these sporidia, placed in water in the morning, presented, at nine o'clock of the same evening, a sprout from one of the extremities, measuring half the length of the spore. In the morning of the next day this sprout had augmented, and become a filament three or four times as long. The next day these elongated filaments exhibited some transverse divisions and some ramifications. On the third day, the germination being more advanced, many more of the sporidia were as completely changed, and presented, in consequence of the elongation, the appearance of a cylindrical ruffle, the cellular prolongations arising from the germination having a tendency towards one of the extremities of the longer axis of the sporidium, and more often to the two opposed extremities, either simultaneously or successively. Out of many hundreds of sporidia examined during germination, he had only seen a very few exceptions to this rule, among which he had encountered the centrifugal tendency to vegetate by two opposed filaments, proving that if it bears a second by the side of the primal filament situated at one of the poles, a second would also be seen from the side of the filament coming from the opposite pole.

Before being submitted to the action of water, the contents of the sporidia seemed formed of two distinct parts, one big drop of yellow oil of the same form as the sporidium, with the space between it and the cell wall occupied by a clear liquid, more fluid and less refractive, nearly colourless, or at times slightly roseate. As the membrane absorbed the water by which it was surrounded, the quantity of this clear liquid was augmented, and the rosy tint could be more easily distinguished. All the contents of the spore, which up to this time remained divided into two parts, presented altogether one aspect, only containing numerous granulations, nearly of equal size, completely filling it, and reaching the inner face of the sporic membrane.

After this time the sporidium augments in size very rapidly, becoming at times irregular, and sometimes even as much as from two to three times its original dimensions, then there appears at the surface, usually at one of the poles of the ellipse, a small prominence, with an extremely fine membrane, which does not appear to separate itself from that which surrounds the sporidium, and it is difficult to say whether it is a prolongation of the internal membrane going across the outside, or simply a prolongation caused by a continuation of tissue of an unique membrane. Sometimes there may be seen at the point where the primal filament issues from the sporidium a circular mark, which appears to indicate the rupture of the external membrane. From this time another change comes over the contents. We again find the yellow oily liquid, now occupying the external position, with some drops of colourless or roseate liquid in the centre, so that the oily liquid and the more limpid fluid interchange the positions which they occupied previous to the commencement of germination. Whether these two fluids have undergone any change in their constitution is difficult to determine, at all events the oily liquid appears to be less refractive and more granular, and it may be that it is a product of new formation, containing some of the elements of the primitive oily drop. Having regard to the delicate character of the membrane of the germinating filaments, De Seynes supposed that it might offer greater facility for the entrance of water by endosmose, and account for the rapid enlargement of the sporidia. By a series of experiments he became satisfied that this was the case to a considerable extent, but he adds:--"I cannot help supposing that a greater absorption of greasy matter in the cell which is the first product of germination raises an objection to an aqueous endosmose. One can also see in this experience a proof of the existence of two special membranes, and so suppose that the germinative cell is the continuation of the internal membrane, the external membrane alone being susceptible of absorbing the liquids, at least with a certain rapidity."

In other Discomycetes germination takes place in a similar manner. Boudier narrates that in Ascobolus, when once the spore reaches a favourable place, if the circumstances are good, i.e., if the temperature is sufficiently high and the moisture sufficient, it will germinate. The time necessary for this purpose is variable, some hours sufficing for some species; those of A. viridis, for example, germinate in eight or ten hours, doubtless because, being terrestrial, it has in consequence less heat. The spore slightly augments in size, then opens, generally at one or other extremity, sometimes at two, or at any point on its surface, in order to pass the mycelium tubes. At first simple, without septa, and granular in the interior, above all at the extremity, these tubes, the rudiment of the mycelium, are not long in elongating, in branching, and later in having partitions. These filaments are always colourless, only the spore may be coloured, or not. Coemans has described them as giving rise to two kinds of conidia, the one having the form of Torula, when they give rise to continuous filaments, the other in the form of Penicillium, when they give birth to partitioned filaments. De Seynes could never obtain this result. Many times he had seen the Penicillium glaucum invade his sowings, but he feels confident that it had nothing to do with the Ascobolus. M. Woronin has detailed some observations on the sexual phenomena which he has observed in Ascobolus and Peziza, and so far as the scolecite is concerned these have been confirmed by M. Boudier.

There is no reason for doubt that in other of the Discomycetes the germination of the sporidia is very similar to that already seen and described, whilst in the Pyrenomycetes, as far as we are aware, although the production of germinating tubes is by no means difficult, development has not been traced beyond this stage.

Seynes, J. de, "Essai d'une Flore Mycologique de la Montpellier," &c. (1863), p. 30.