Immunity in infective diseases

For several reasons this immunity in the vegetable kingdom cannot be treated in a satisfactory fashion. Much attention has been devoted to the pathology of plants and the etiology of a number of vegetable diseases was well established at a period when we were still groping in the dark for the causes of infective diseases in man and the higher animals. In spite of this, the botanist has relegated the study of the phenomena of immunity to a secondary position, and up to the present no work specially devoted to this subject has appeared. It is only incidentally that the question of the resistance of certain plants to morbific factors capable of infecting or intoxicating them has been touched upon. We should require, therefore, to carry out special researches in this direction and to make a very complete study of botanical literature, before we should be able to present to our readers a résumé of the question of immunity in the vegetable kingdom. Such a programme being impossible we must content ourselves with borrowing from the botanists certain facts which throw light on some aspects of the general problem in which we are interested.

Many of the higher plants are subject to infective diseases set up by the lower plants, of which the most important are the Fungi. Whereas in the animal kingdom the majority of the infections are due to Bacteria, these micro-organisms rarely occur in plants; moreover when they are present the part they play is nearly always a secondary one. This difference is due mainly to the chemical composition of the “humours” in the two kingdoms, the cell-juice of plants being generally acid; under this condition the Fungi develop much better than do the Bacteria.

The various modes of defence against infective diseases that have been met with in unicellular organisms are also found in the multicellular plants. Whereas in almost all plants the cells are rigid, owing to the presence of a well-developed membrane, some of the lower plants have preserved a condition in which the protoplasm is completely naked and capable of movement. Myxomycetes are specially distinguished by an amoeboid stage of existence and by the formation of large plasmodia which protrude protoplasmic processes and exhibit a kind of locomotion similar to that met with in the Rhizopods and the Sporozoa.

Infective diseases among the Myxomycetes must be very rare since, up to the present, they have not been noted by a single observer. It is very probable that the plasmodia get rid of the infective germs, as do the Protozoa, both by expulsion of the parasites and by means of intracellular digestion. This latter takes place in a medium which is distinctly acid and by means of a soluble ferment described by Krukenberg^[37] as a kind of pepsin. I need not here enter into further detail as I have already treated this subject in my Lectures on the comparative pathology of inflammation. The fact that the Myxomycetes can ingest living organisms has been demonstrated by Celakovsky, jun.^[38], who has observed that the spores of the various Fungi can germinate in the interior of the plasmodium. Whilst our conceptions concerning the resistance of the plasmodia in regard to micro-organisms are merely based upon analogies and hypotheses, our ideas as to their immunity against soluble substances rest on well-established experimental facts. We owe to Stahl^[39] our first information as to the mode by which the plasmodia resist poisons. When they are placed in contact with solutions of salts, of acids or of sugar in a sufficiently concentrated form to bring about an injurious action, the plasmodia make use of their amoeboid power of motion to escape from these fluids. Hence they exhibit a negative chemiotaxis, exactly parallel to that so often observed in the case of the unicellular organisms. Consequently there is in the Myxomycetes a natural immunity due to the activity of their movements. Further, a kind of acquired immunity in these plants has also been demonstrated by Stahl. The following is the passage in his paper referring to this subject, a passage very important from a general point of view^[40]: “If we replace the water in a vessel by a 1 or 2% solution of glucose, we observe either the death of the plasmodia, if the action is too rapid, or merely their retreat from the glucose solution. Even solutions of ½ or ¼% are at first avoided by the plasmodia and, should the action be too rapid, may cause their death. Usually the plasmodia emigrate into those portions of the substratum remote from the solution, to return after some time, often only after several days, and immerse themselves in the solution of glucose as they do in an infusion of tan, although with more hesitancy. Consequently the Myxomycetes accommodate themselves slowly^[41] to a more concentrated solution, probably by giving up a certain proportion of their water. I was able to observe the same phenomena with even much more concentrated solutions (2%). A plasmodium which at the end of several days had adapted itself to a 2% solution of glucose and had sent out numerous processes into it, found itself injuriously affected when the sugar solution was suddenly replaced by pure water. Those that remained alive had retired to a great distance from the upper layer of the fluid and did not descend again until the end of the second day. After a fresh change of fluid we were able to observe first the repulsion and later the attraction of the plasmodia, but a certain time always elapses before the plasmodia become accustomed to the change in concentration. We obtain the same result when we replace a 2% solution, not by pure water, but by a ½ or a 1% solution” (p. 166).

De Bary^[42] had already interpreted these facts as being due to an immunity acquired by the plasmodia, the result of an adaptation of these organisms to solutions which they had, at first, carefully avoided. He threw out the suggestion that a similar adaptation might take place in regard to solid substances ingested by the Myxomycetes.

As these phenomena of acquired immunity, in organisms so primitive and of so simple a structure, are of immense importance from the point of view of Immunity in general I felt bound to submit them to a personal investigation. I found it an easy matter to accustom the plasmodia of Physarum to solutions of arsenious acid which at first repelled them in a very striking manner. This adaptation manifests itself by movements of the plasmodia and by the change from negative chemiotaxis (repulsion) to positive chemiotaxis (attraction).

It is impossible in the present state of our knowledge to state precisely the modifications that the plasmodia undergo during this process of adaptation. Stahl supposes that they depend “on some special properties of the plasmodia (probably in a greater or less richness in water)”; and that it is a case “not of simple phenomena, easy of explanation, but of extremely complicated phenomena of irritability.”

It is evident that, in this case of acquired immunity, we have not to do with a question of physical or chemical modification of the solutions employed but solely with reactive phenomena on the part of the living plasmodia.

After a phase of active life, during which the Myxomycetes move, feed, digest and expel waste products as do the lower animals, there comes a stage when they become immobile and transform themselves into a number of sporangia filled with rounded spores. Before leaving their animal aspect for that of true plants, the plasmodia exhibit entirely new attributes. They reject all nourishment and no longer ingest foreign bodies; they avoid the moisture which previously attracted them and cease to shrink from the light.

Having come to maturity, the Myxomycetes declare themselves true plants and lead a passive life until the new generation comes forth. Most plants are restricted to this passive phase of the Myxomycetes. In these latter it persists only for a short period, whereas in almost all plants it is the permanent condition. It is at this stage that these organisms are liable to the attack of parasites against which it is necessary for them to oppose all their means of defence. Our knowledge of these means of defence is as yet, as I have already stated, very imperfect, and the example of Sclerotinia libertiana (or Peziza sclerotiorum) which has been the subject of the researches of de Bary^[43] remains up to the present the one that has been most thoroughly studied.

This Fungus, belonging to the group of the Discomycetes, invades many species of plants and often produces great ravages amongst the cultivated plants of our fields and gardens, such as colza, hemp petunias, dahlias, etc. The mycelium of this Sclerotinia develops in the stems of herbaceous plants and produces sclerotia inside them, forms of resistance, which in this instance are black and resemble small particles of mouse excrement.

The spores of the Sclerotinia germinate and form mycelial threads on the surface of the plants. In order that they may penetrate into the tissues these threads must attack the cell-membrane and for this purpose they secrete a fluid, which contains both a digestive ferment and oxalic acid, the latter being essential for the action of the ferment.

The presence of this “toxin” has been demonstrated by de Bary by macerating the mycelium of the Sclerotinia. The resultant extract has a well-marked action on the tissues of many plants (carrot, Jerusalem artichoke, chicory, etc.). Under its influence the protoplasm of the cells contracts, a genuine plasmolysis is set up, the cell-membrane swells and its layers between the cells are dissolved. As the result of this digestive action, the cells become separated and the tissue softens. This extract, when heated to 52° C., loses its digestive action on the cellulose membrane, but still retains its power of setting up plasmolysis. This reaction to temperature confirms the view that the juice of the Fungus contains a soluble ferment. The results of de Bary’s researches have been confirmed and in part supplemented by the experiments of Laurent^[44].

It is a fact of common observation that the Sclerotinia libertiana invades for the most part young plants. It may therefore be asserted that the disease produced by this Fungus is, like scarlatina or measles in the human subject, an “infantile” disease. De Bary suggested that the immunity of adult plants must depend on the greater resistance which their cell-membranes offer to the fluid secreted by the mycelial filaments. Direct experiments have shown the accuracy of his suggestion. Whilst the fluid extracted from the Sclerotinia readily digests the tissue of young plants it leaves intact that of adult plants of the same species.

In the course of this disease we have a struggle going on between two plants. The parasite brings into play toxic and digestive secretions with which it seeks to impregnate its host. The attacked plant defends itself by the secretion of membranes capable of resisting the action of the secretions of the Fungus. This struggle by means of chemical substances is, however, directed by the activity of the living cells of the two belligerent plants, an activity dependent upon the irritability of their protoplasm.

The example we have just studied may serve as a type for our examination of the phenomena of immunity in the vegetable kingdom. The crux is above all to prevent the access of the parasites to the vital parts of the plant by opposing to them membranes as resistant as possible. Consequently the majority of plants, directly the smallest lesion is produced, react by an abundant cell-proliferation and by the suberisation of the outer layers. The cell-membranes of the latter thicken, the cellulose is transformed into suberin; a layer of cork not very permeable to fluids and gases being thus formed. By suberisation the plant reacts against grosser lesions, incisions or burns, as well as against the decay set up by micro-organisms.

Massart^[45], in an extremely interesting memoir, has brought together the known data concerning cicatrisation in plants and has demonstrated the fact that it is a very variable process. In many leaves after being damaged there is no attempt to react by forming cicatricial tissue. Many aquatic and marsh plants react but feebly. Their tissues die and turn brown, the plants failing to defend themselves by cicatrices, probably owing to the ease with which the lost parts can be replaced. When, however, in the same plants, there is produced a lesion of parts which are of great importance for the preservation of the integrity of the individual or a lesion of the organs which enable the plant to continue its existence through the winter, cicatrisation of the wounds takes place rapidly.

The old or adult parts in most cases react differently from the young parts. Thus, the young leaves of Clisia (the example selected by Massart) react to traumatism very promptly and form a genuine callus which makes good the injury, but the adult leaves merely produce a layer of cork in the immediate neighbourhood of the lesion.

The essential mechanism of cicatrisation has not yet been satisfactorily analysed, but it is evident, when all is said and done, that it is directed by the irritability of the living protoplasm of the vegetable cells.

Many plants protect their wounds with a kind of dressing, using for that purpose juices which harden on exposure to the air. Sometimes these juices, e.g. latex, are preformed in the plant and are as it were always ready for use; at other times they may be formed only as the result of an injury. In this latter case the resins and gums which serve to close the wound and to protect the living parts receive the name of “cicatricial secretions” (Wundsecrete). According to the view first formulated by de Vries, those juices which harden under the action of air prove of great service both as natural “dressings” and as safeguards against the attacks of plants and animals. Indeed many of these secretions contain essences whose antiseptic and toxic action is now generally recognised^[46].

The suberisation, the formation of a callus, and the secretion of juices which close the wounds, are all means readily utilised and very potent in ensuring the resistance of plants against all sorts of injurious influences which may be set up by a morbid condition. But these processes are not the only means which plants have at their disposal. The living elements of plants usually secrete a cell-juice of acid reaction which plays a very important part in the defence of plants against pathogenic agents. Laurent^[47] has studied this phase of the immunity of plants against bacterial decay. A variety of the Bacillus coli communis, according to this observer, attacks the potato by means of its secretions in a fashion analogous to that already described when discussing Sclerotinia. This bacillus produces a soluble ferment which has the power of digesting the cellulose membrane in the tuber of the potato, and at the same time secretes an alkaline juice without which this digestion cannot go on. Heating to 62° C. destroys the soluble ferment and the fluid thus heated is no longer able to digest the layers of the cell-membrane between the cells. In spite of exposure to this temperature, however, it still retains intact one or even several substances which may continue to cause contraction of the protoplasm and ultimately kill it.

When Laurent placed cut halves of tubers coming from races of potato which were most resistant to bacterial decay in the fluid produced by the Bacillus coli and afterwards inoculated them with the bacillus itself, he invariably found that the vegetable cells were profoundly affected.

The alkaline secretions of the bacillus studied by Laurent may be neutralised by the acid juice of the potato, and when certain races of tubers prove immune from decay, it is, according to this observer, because of the production of sufficiently acid cell-juices. Moreover he actually succeeded in communicating an artificial immunity to varieties of the potato which were most susceptible to decay by immersing them for several hours in solutions of certain organic acids. On the other hand, when he treated varieties endowed with a well-marked natural immunity with alkaline solutions, the tubers became very susceptible to the decay set up by the bacillus.

The struggle between the potato and the Bacillus coli reduces itself, then, to the chemical reaction between the alkaline cell-secretions of the micro-organism and the acid secretions of the potato. This general fact, according to Laurent, explains the part played by certain manures in determining the susceptibility or the resistance manifested by the potato and many other plants against infective diseases.

We know that the addition of phosphates to the soil increases the immunity of certain cultivated plants. These substances are greedily absorbed by the roots and produce acid salts which are dissolved in the cell-juice. The nitrogenous manures, on the other hand, both potassic and lime, diminish the resistance of the same plants, probably from the fact that they bring about a diminution of the acidity of the cell-juice.

But these manures can act differently on different plants. Thus the same phosphates which confer immunity on the potato against bacterial decay render the Jerusalem artichoke more susceptible to the attacks of the Sclerotinia.

Laurent explains this fact as due to the difference in the reaction of the medium, which favours the action of one or the other of the soluble ferments of the two parasites. The ferment of the bacillus digests the cell-membrane in an alkaline or feebly acid medium, whereas the hyperacidity which results from the absorption of the phosphates prevents this digestion and consequently aids the plant in its struggle. On the other hand, the ferment of Sclerotinia, as is seen from the researches of de Bary, will digest cellulose even in a distinctly acid medium. The hyperacidity, induced by the phosphated manure, in this case favours the parasite and enables it to gain the upper hand in the struggle with the tissues of the artichoke.

In addition to neutralising the microbial products the acids of the cell-juice also act injuriously on most bacteria, which will only develop in neutral or alkaline media; it is for this reason that bacterial diseases are so much rarer in plants than in animals.

The secretion of cell-juices is consequently a very important element in the defence of plants; it will be useful, therefore, to ascertain as definitely as possible the essential mode of its action. Vegetable cells are as a rule very sensitive to the influences to which they are exposed; they distinguish with great precision the changes which take place in their surroundings. They are, indeed, capable of discerning not only the physical properties but also the chemical composition of the medium in which they live.

Vegetable cells estimate very accurately the osmotic pressure of the fluid which bathes them, and they react towards this solution by increasing or diminishing their own internal pressure. Van Rysselberghe^[48], in an investigation very carefully carried out, demonstrated that when vegetable cells (especially the epidermic cells of certain species of Tradescantia) are placed in a solution of greater density than that to which the cells are accustomed, the intracellular pressure increases; in a solution of less density the pressure diminishes. These changes in osmotic pressure are due to variations in density of the cell-juice, whilst these variations are in turn determined by chemical transformations. Thus, if the cell be treated with a too concentrated solution it produces oxalic acid, which dissolving in the cell-juice, is, owing to the smallness of its molecule, very osmotic.

With the purpose of confirming this by exact facts van Rysselberghe has studied the acids of the cell-juice of Tradescantia. In the normal juice he found that malic acid was constantly present and, in rare cases only, traces of oxalic acid. He then determined the acids present in the leaves of the same plant after they had been several days in contact with fairly concentrated solutions of cane sugar. In each analysis he found oxalic acid in quite appreciable quantity. There is then, in the plant which adapts itself to more concentrated solutions of the medium, a production of oxalic acid which serves the purpose of increasing the pressure of the cell-juice.

The origin of this oxalic acid could not be accurately demonstrated, but van Rysselberghe considers that it is probably formed at the expense of the glucose.

According to the researches of Giessler oxalic acid is localised specially in the epidermis and generally in the peripheral tissues of plants; it is very probable, therefore, that it fulfils a protective rôle against all kinds of injurious influences. Botanists hold indeed that oxalic acid keeps herbivorous animals, especially slugs and plant lice, from attacking plants that are rich in this substance. It is of use, also, in retaining the moisture in the superficial cells. It is very probable that it also plays an important part as a factor in the maintenance in plants of immunity against bacterial diseases.

The vegetable protoplasm, which is capable of increasing the production of acids in order to raise the osmotic pressure, can also, in case of need, cause a diminution.

When the cells of Tradescantia are transferred from a concentrated solution into one much more dilute there may often be observed a precipitation, in the cell-juice, of crystals of oxalate of lime; this brings about a diminution in the osmotic pressure. When the density of the medium is altered, and the vegetable tissue is again transferred to a stronger solution, the oxalate crystals are seen to dissolve, as a result of a new production of acid.

These chemical processes, so important to the life of plants in general and for ensuring to them immunity against infective agents in particular, are dependent upon the irritability of the protoplasm. Imprisoned in its resistant and more or less thick membrane, the living part of the vegetable cell estimates with nice discrimination every change that takes place around it.

Massart^[49] has shown that the stimulation produced by traumatism is often propagated a considerable distance and may excite a reaction in very remote cells. If the mid-rib of a leaf of Impatiens sultani be cut near the base of the limb the wound does not cicatrise but, a few days later, the leaf becomes detached from the stem.

Irritability is a fundamental property of all living beings. The plant may react by rapid movements, as in the case of the Mimosa pudica, or more slowly—by chemical reactions—as in the case of adaptation to density of medium. These reactions are produced as the result of various irritabilities which exhibit a specific character.

It is this specificity that determines whether the reaction that is manifested by the movements shall be produced in this direction or in that. The stem, owing to the specific irritability of its living parts, turns to the light; whilst the root, guided by a different irritability, grows down into the soil.

The irritability of plants, like that of unicellular organisms, is subject to the psycho-physical law of Weber-Fechner. Pfeffer^[50] first demonstrated this for the motile spermatozoids of the Cryptogams. Massart^[51], by a series of ingenious experiments on the irritability of a Mould (Phycomyces nitens) to light, has shown that the same law regulates the movements of this plant towards the source of light. This irritability of the Fungus to light is much more delicate than is the chemiotaxis of the spermatozoids of the Mosses and the Ferns and than that of the Bacteria.

Errera concluded from a consideration of the experiments of van Rysselberghe that the osmotic reaction of plants must also come under this psycho-physical law. His pupil at his request made systematic researches on the subject and the results have entirely confirmed his prevision. According to the data obtained by van Rysselberghe^[52], the cellular osmotic reaction increases in arithmetical progression as the osmotic stimulation increases in geometrical progression. The osmotic reaction is therefore proportional to the logarithm of the stimulation.

To sum up, the phenomena of adaptation and of immunity in plants are, as in the unicellular organisms, very widely distributed. Plants defend themselves by means of their resistant membranes and by secretions whose physical and chemical properties they are able to modify. These phenomena are dependent on the living parts of the cell which regulate them according to their greatly developed irritabilities. Thanks to this power, plants can gradually adapt themselves to concentration of the medium and to the presence of poisons which, at first, set up serious disturbances. Plants therefore, alongside a natural immunity, possess an acquired immunity against many pathogenic agents.