Crown Gall-a Malignant Growth

A. J. Riker, A. C. Hildebrandt.

Crown galls are diseased growths that occur on peaches, apples, raspberries, roses, sugar beets, and a great many other broadleaved plants. The galls appear commonly where the plants come out of the ground, the crown hence the name.

They ordinarily are quite soft. They have neither a definite exterior like a bark layer nor a woody interior like a stem. Having no protection against secondary invaders, the galls become hosts to various bacteria, fungi, and even insects, particularly during wet weather. A type of decay like soft rot sets in.

A gall contains a disorganized mixture of large, swollen cells; small cells that divide rapidly; and sap-conducting cells, which have a ladder-like thickening in the walls. The gall may seem hard if the woody cells are abundant.

The nonparasitic bur knots, callus overgrowths, and infectious hairy root are common diseases that have been mistaken for the true crown gall.

The disease occurs the world over. Infected nursery stock easily could have carried it from one place to another. Its economic importance varies. In irrigated districts and other sections with abundant moisture, the disease may occur so often that an uninfected plant is hard to find. A gall that develops on a lateral root may cause little damage. A gall that occurs on the main stem near the crown and involves a considerable part of the circumference of the stem, may weaken the stem, disrupt the flow of sap, and favor the progress of a cortical rot. Such a plant usually dies.

Crown gall is caused by the bacterium Agrobacterium tumefaciens, a small Gram-negative rod. It is closely related to the bacteria that produce root nodules on leguminous plants and to bacteria of the colon-typhoid group.

The crown gall bacteria grow readily on any of the common bacterial media. They do well on nitrate-sucrose-mineral salt agar.

The infection cycle is relatively simple. The bacteria enter the tissues apparently only through wounds, most commonly through wounds caused by insects or cultivation. Once inside the tissue, they occur primarily between the cells, from which they stimulate the surrounding cells to divide. In the earlier stage, that looks like the response to a wound, but it never heals. As the galls increase in size, some of the larger cells apparently are crushed by the pressure, and the bacteria move into other tissues for further activity. The bacteria occur usually in abundance on the surface, from which they may be washed off and distributed by flowing water. Chewing insects may carry them from one plant to another and also may introduce them into wounds. Over long distances the bacteria travel on the surface of nursery stock or inside the tissue. Symptoms may not develop for several weeks, depending on temperature, humidity, and the growth of the host. They may not show during nursery inspection.

Galls ordinarily develop better as the temperatures increase up to a certain point. However, on tomato, Kalanchoe, and certain other plants studied in experiments, the galls fail to develop much, if any, above 83 F., although plants and bacteria do well at the higher temperatures.

Moisture, light, and mineral nutrients may influence the development of the galls. Frequently, since no growth means no gall, that merely reflects the growth of the plant itself.

Various insects living in the ground seem important, especially those on raspberries. They chew on the roots and the galls. They open infection courts and may actually transmit the bacteria from one injury to another. Cultural practices likewise may be important. Obviously a type of cultivation that encourages insects or produces many injuries on the roots or crowns may encourage infection.

The means for combating crown gall are closely tied in with the environment and the way the disease develops. Perhaps one of the best control measures is to grow a crop that is not susceptible for several years between crops that are susceptible. A crop that reduces the presence of root-chewing insects likewise discourages this means of transmission. Sometimes, if the infection is carried on the surface of the planting stock, a surface disinfectant may be helpful but not fully reliable, because in some instances the bacteria may enter a wound and be protected against the disinfectant. Such infections are impossible to detect during nursery inspection because frequently the period of incubation is not long enough to permit gall development. Galls that develop in the nursery on the unions of piece-root apple grafts have been controlled by special adhesive tape wrappers. They may contain the disinfectant, corrosive sublimate, in the adhesive mixture.

WHILE THE ECONOMIC importance of crown gall makes it a critical disease, particularly on sugar beets, fruits, and some types of nursery stock, it has still greater importance as a tool for work dealing with the fundamentals of diseased growth. Erwin F. Smith called crown gall a "plant cancer."

The changes from normal growth to diseased growth involve many fundamental biological problems. The more one learns about biochemistry the greater appear the parallels between Plants and animals between cabbages and kings. From the standpoints of growth stimulation and, what is much more important, growth inhibition, many basic substances, including various carbohydrates, fats, proteins and their derivatives, mineral salts, vitamins, and enzymes, are common factors occurring both in plant and animal cells. Admittedly, a description of such fundamental work becomes a bit technical. Its importance extends far beyond the agricultural field.

For fundamental work on growth, plants have certain advantages over animals. The plants have no complex nervous, digestive, and circulatory systems, which complicate the basic physiology. Large numbers of plants may be used at a relatively low cost. The possibilities for genetic purity with plant material are real and important. Many inbred lines are available for use. Still better, various plants, such as many fruits, are ordinarily reproduced by vegetative propagation. Thus, the different individuals are genetically identical. For details of tissue metabolism, tissue cultures from higher plants offer a relatively simple and direct approach not yet possible with tissue from higher animals. These cultures grow indefinitely upon media which contain only nutrients with known chemical formulas. Ordinarily the tissues grow well without a change of nutrient for some weeks. The growing tissue develops in a compact mass easily separated from the medium. Thus, any change in growth may be determined merely by weighing the tissue pieces. Many ways are available for inducing at will one or another kind of diseased growth.

What actually initiates these diseased growths, what keeps them going, and especially how they can be inhibited are critical questions. They have stimulated much speculation and many experiments. One may approach this problem from the standpoint that the bacteria start off the diseased development. They may or may not be necessary to keep the diseased growth going. To make a comparison with firearms, one might consider that the causal agent operates as a trigger mechanism to set things off. However, a trigger alone is not enough. The gun must be loaded. Also important are the amount of the load, the character of the load, the amount of dampening the load carries, and so on.

Detailed data on the metabolism of the plant, of the causal agent, and of both together are necessary.

As the crown gall bacteria develop in suitable culture media, a number of physical and chemical changes occur. Knowing what happens in such media may help to clarify the action of the bacteria as they work in host tissue.

Among the critical physico-chemical changes are the modification of the hydrogen-ion concentration, a reduced oxidation-reduction potential, a decreased osmotic pressure, and an increased viscosity.

Among the chemical factors are the ability to use an unusually large number of different sources of carbon and nitrogen. Likewise these bacteria tolerate many kinds of inhibiting substances.

The metabolic products known to be formed by crown gall bacteria have thus far been surprisingly simple, principally carbon dioxide. No volatile organic material has been detected. By far the most common residual metabolite is a bacterial gum. In Culture its weight is considerably greater than that of the bacterial cells. One molecule of this gum contains approximately 24 glucose molecules. The gum is viscous, takes up moisture, and is chemically rather inert. Apparently neither the bacteria nor the host plant has an enzyme system capable of attacking it.

The metabolites resulting from nitrogen in the medium have received much less attention. Ammonia was one of the first products reported and has come the nearest to being a common factor of any found among the various cell-stimulating bacteria.

The crown gall bacteria have been shown to produce the vitamins biotin, riboflavin, pantothenic acid, and thiamine. Since they grow in synthetic media, they produce any other such material needed for their metabolism.

An analysis of large quantities of the crown gall bacteria has revealed the presence of various lipids. These are more or less toxic when strong preparations are placed upon host plants.

The attenuation of the virulent culture by means of certain amino acids and related compounds has an interesting bearing upon this problem. The cultures were grown in media with a relatively alkaline reaction and only a tiny bit of the amino acid, glycine, but enough to reduce but not to stop growth. After a series of 15 or more successive transfers made at intervals of several days, the cultures gradually lost their capacity to induce gall formation. In some cases, if the attenuation was not carried through too many transfers, virulence was restored by a series of transfers on ordinary media. In other cases where the cultivation on glycine was carried several transfers beyond the point of attenuation, such restoration did not occur in 4 years.

A restoration of virulence has been accomplished also by irradiating partly attenuated cultures with ultraviolet light so as to kill all but one in a thousand. The survivors commonly showed a conspicuous increase in virulence.

The morphological responses of the plant tissue to crown gall bacteria show important changes. The wound that introduced the bacteria flooded the intercellular spaces and provided a culture medium for the bacteria. The cells around the bacteria enlarged within 2 days and the adjacent cell walls turned somewhat brown and took ordinary stains more intensely than normal walls. Within 4 days new crown gall cells had formed. In the early stages of development the new cell walls were laid down in somewhat the same manner as those from a wound.