Thermal studies on the factors responsible for tumor initiation in crown gall.

ABOUT TWENTY years ago Riker (1926) reported the results of a study which dealt with the influence of temperature and soil moisture on the development of the crown-gall disease in tomato plants (Lycopersicon esculentum Mill.). These experiments demonstrated that good tumor development was obtained at temperatures up to 280C. The tumors were found to be distinctly inferior between 280C. and 300 C., while above 300C. no tumors were produced. This was true despite the fact that both the host plant and the inciting agent of the disease, Phytomonas tumefaciens (Smith and Town.) Bergey et al., grew well at 320C. In a more recent contribution on this subject Riker, Lyneis and Locke (1941) inoculated, in addition to tomato, a number of other hosts. They found that some species of plants tested behaved like tomato and produced tumors only at 300C. or below, while certain of the Nicotiana species produced galls both below and above the critical temperature for the tomato. The tumors that developed at 31 0C. were smaller, however, and made their appearance from a week to 10 days later than those produced at 26?C. On the basis of these results it was concluded that ".... the effect temperature has on pathogenicity seems correlated more with the host than with the bacteria." These observations clearly indicate that a pronounced change in the physiology of either the host or the bacteria, or perhaps both, occurs between 28?C. and 31C. The findings, therefore, suggested a useful experimental approach for further study of the conditions under which the factors responsible for tumor initiation in crown gall are physiologically active in altering the normal host cells to tumor cells. The results reported below are concerned with that phase of the crown-gall problem. EXPERIMENTAL METHODS.-Preliminary experiments demonstrated that the inoculated periwinkle plant (Vinca rosea L.) does not produce tumors at or above 31 C. In its reaction, therefore, it was similar to the tomato. The periwinkle, however, has a great advantage over the tomato as an experimental subject because, as Braun (1943) has shown, it can be freed of the inciting agent bv thermal treatment at any desired time without altering the capacity of the tissues to produce typical tumors at the points of inoculation. By eliminating the organism it was, of course, possible to study the host reaction without concern for recurrent injury and resultant reinfection. The method of inoculation of the periwinkle and the thermal inactivation of the organism was the same with one exception as that described earlier by Braun (1943). The heat-treated plants were 1 Received for publication December 20, 1946. kept at 46?C. for 3 days instead of the 5 days previously suggested. This period was found to be adequate for the complete elimination of the crowngall organism from the host. A thermostatically controlled chamber having a temperature of 32?C.+.5?C. was used throughout the work. During the warm summer months the chamber was kept in a dark room having a temperature of about 200C. It was illuminated 15 hours daily with six 350-watt lamps. During the cooler months the chamber was placed on a greenhouse bench with no artificial illumination. The relative humidity of the chamber throughout the year was about 90 per cent. All experiments described below were performed on at least two different occasions unless otherwise noted. EXPERIMENTAL RESULTS. Braun and Laskaris (1942) have suggested that there are at least two distinct phases involved in the mechanism of tumor formation in the crown-gall disease. In the first phase the normal host cells are changed into tumor cells which, without stimulation, will not develop into a neoplastic growth. The second phase, according to this concept, involves those factors that are concerned with the initiation of growth anid the continued multiplication of the altered cells. The need for recognizing a distinction between those factors that render cells neoplastic and those that affect their subsequent behavior was further enmphasized by Braun (1943). It was necessary at the outset, therefore, to determine whether the temperature of 320C., which is just above the critical temperature for tumor formation, prevented the conversion of normal cells to tumor cells or exerted an inhibitory influence on cellular multiplication after the change had been consummated. In order to study this question two sets of large, woody periwinkle plants, consisting of four plants each, were inoculated with the Brown peach strain of Phytomonas tumefaciens. One hundred or more punctures were made in each plant. Following inoculation one set of plants was placed immediately in a chamber at 32?C. while the second set of four plants was kept at a temperature of 26?C. Both groups were incubated for 5 days at their respective temperatures. A 5-day period of incubation was used in this study because it had previously been shown (Braun, 1943) that the cellular alteration was fully accomplished in that period in Vinca plants. All plants were then placed in a hot room having a temperature of 46?C. for 3 days. This was done to free the plants of Phytomonas tumefaciens. Following the heat treatment, that group of plants which had been incubated at 260 C. was