The old ideas about continuing diseased growth without the causal agent, once disease in the tissue was started, thus have received experimental evidence with periwinkle. This brings closer still the comparison between crown gall and certain diseased growths in animals. How frequently such autonomous growth also occurs in other plants remains uncertain. The failure of many "secondary" crown galls on sunflower, Paris-daisy, and marigold to continue development after the bacteria have died has suggested caution about too broad inferences.

The differences in chemical compositions between the galls and correspondingly healthy tissue have been examined. Approximate analysis indicated that the gall tissues resemble those of young plants, being high in nitrogen and low in fibrous material. Considerable variations were observed, depending on the time of collection and on the species of plant. The actively growing tissues contained more ascorbic acid. An increase of thiamine appeared in the gall tissue within a week.

The enzyme content of the galls was different from that of the healthy tissue. The galls contained 86 and 57 percent more than the stems of catalase and peroxidase on a total nitrogen basis. The galls had active tyrosinase, while the stems had little, if any. Furthermore, the galls contained relatively much more glutathione than the stems.

Reduced respiration levels by the gall tissues have appeared important. With the considerable excess of oxidizing enzymes, the suggestion has appeared that the basic metabolic activity of the galls may be relatively anaerobic in comparison with that of the neighboring tissue. This condition deserves further study as a causal factor for cell stimulation. It may be correlated perhaps with the earlier observations that the tissues involved had flooded intercellular spaces. The diffusion of oxygen through normal tissue is 2,000 to 3,000 times greater than that in tissue with water in the intercellular spaces.

As the amount of hyperplastic tissue increases in size, more difficulty with gas exchange doubtless also develops. The crown gall bacteria lower the oxidation-reduction potentials of the material in which they are growing. The formation of ammonia and the consequent change of the pH in an alkaline direction also lowers the oxidation-reduction potential. The bacteria and gum block certain intercellular spaces. The presence of gummy materials, which are hygroscopic, might cause the cells to swell and to metabolize more slowly. The reduced oxygen uptake in the presence of 3-indoleacetic acid at a gall-inducing concentration has added interest.

Among the well-known growth substance effects are increased epinasty, adventitious roots, cambial activity, bud inhibition, and delayed abscission. All were found associated with crown gall on tomato. The presence in galls has been amply demonstrated of something like 3-indoleacetic acid at approximately 6 to 12 parts per billion an amount comparable to that in some actively growing and normal plant parts. While these extremely small amounts strikingly affect certain tissues, much stronger and almost lethal concentrations are needed to induce chemical galls.

The stimulation of tissue about inoculations with the attenuated crown gall bacteria has been possible not only with the virulent culture higher on the stem but also with galls induced by certain chemicals. However, no correlation has appeared between the formation of these chemical galls and any other growth substance effects.

The possibility of studying the physiology of these diseased tissues has been greatly enhanced by the development of tissue cultures. These consist of masses of largely undifferentiated callus that grow indefinitely on synthetic media. Thus, science has an important tool to determine which substances the tissues can use, which are not available, and particularly which are inhibiting.

The diseased tissues in culture have been derived commonly from crown galls that were free from bacteria, or from growths having a comparable origin. Extensive studies have been made in relation to the best physical conditions, the importance of plant and bacterial products, the influence of different concentrations of various mineral salts, the responses to the more common growth substances, and the activity of different sources of nitrogen and different sources of carbon the latter including sugars and polysaccharides, alcohols, and the salts of organic acids. Some of the common metabolites appear particularly important, either for the stimulation or inhibition of growth. Likewise, the concentrations of certain metabolites seem as important as the substances themselves.

These tissue-culture studies also have shown some striking differences between the tissues from different species. Are they opening further the door for physiological as well as morphological understanding of tissues? In any case, many interesting possibilities appear for studying various aspects of tissue metabolism in health and disease, for clarifying the relations between host and pathogen, and especially for learning more about diseased growths.

Growth inhibitions from certain amino acids and organic acids have led to the hope that still other and more powerful agents would soon be found. The most active inhibition came with analogs of pteroylglutamic acid. These inhibited callus growth at 10 to 100 parts per billion. The 4-amino-Niornethyl-pteroylglutamic acid applied locally to young crown galls on sunflower at 0.1 milligram per milliliter completely inhibited gall development. Certain nitrogen mustards, penicillin, 8-azaguanine, and cortisone also have inhibited crown gall.

Many trigger agents have been associated with diseased plant growths. Whether they operate by providing stimulation or by removing inhibitors remains to be determined. However, as mentioned earlier, the trigger is not enough. What happens depends on the kind of load and the amount and whether it is dampened by inhibitors. The use of tissue cultures has opened the way for determining much about the load, about dampening materials, and about the amounts of both necessary for effectiveness.

Basic information is being developed not only for the influence of common mineral salts, sources of carbon, sources of nitrogen, and various metabolites, but also for concentrations that encourage or inhibit growth.

The idea that diseased growth develops from a lack of balance among critical factors fits well into this concept. While we shall continue to analyze individual factors that by their presence or absence may change normal into pathological growth or keep it going, no one thing may be responsible. For normal growth, a number of factors operate in a suitable balance. However, in pathological growth of one kind a group of these factors may be out of balance. Likewise, in pathological growth of another kind, the balance may be disturbed in some other way.

A. J. RIKER has been professor of plant pathology in the University of Wisconsin since 1931. He is the author of Introduction to Research on Plant Diseases (with R. S. Riker) and many research reports on bacterial plant diseases, diseases of forest trees, and factors that influence pathological growth. For 7 years he was an editor of Phytopathology.

A. C. HILDEBRANDT is an assistant professor of plant pathology in the University of Wisconsin. After earning his doctorate at Wisconsin in 1945, he has been engaged in research on crown gall, mineral and carbohydrate metabolism of tissue cultures, environmental factors affecting plant tissue culture growth, vitamins, and growth-regulating substances.