Studies on the Increased Respiration of Potato-Tuber Tissue after Infection with Gibberella saubinetii (Mont.) Sacc.

I The respiratory increase observed in many infected plant parts might be partly or wholly attributed to the gas-exchange of the pathogen. With potato-tuber tissue infected with Gibberella saubinetii (Mont.) Sacc. investigations were made in order to find out whether a respiratory increase could be demonstrated in the host cells, and whether this increase was accompanied by a qualitative change of the respiratory pathway. In the second place the mechanism by which the respiration rate is regulated was investigated. II As the fungus only penetrates the outer cell layers (2-3 mm.) of potato-tuber tissue, the respiration of host cells which did not contain mycelium, could be investigated in the tissue next to the invaded parts. From the two halves of each tuber, one of which was inoculated on the surface of the cut while both were incubated at 25° C., tissue samples were cut at various distances from the surface of the original cut. In the infected halves the respiration was distinctly accelerated in the proximity of the area invaded by the fungus, and decreased with increasing distance to this area, to reach a nearly constant level in the zones at greater distance. Occasionally a zone with soft tissue was found close below the area with mycelium. The 02-uptake of this soft tissue was lower than that of the adjacent apparently normal tissue, the respiration of which was accelerated. The results of many experiments suggested that in tuber halves without a zone with soft tissue differences in respiratory increase and in the distance from the surface where this increase could still be demonstrated, corresponded with differences in intensity of infection and in duration of the incubation at 25° C. (Fig. 2 and 3). In the non-infected halves an increased respiration was observed in a narrow area (2-3 mm.) immediately below the surface of the cut, which was blocked by a cork layer. Ill In order to find out whether the respiratory mechanism had changed qualitatively after infection, R.Q_.-values were determined and the effect of respiratory inhibitors was examined in tissue from non-infected and infected tuber halves. The R.Q_.-values of non-infected tissue and those of host cells which were not penetrated by the mycelium, were equal. The 02-uptake of all samples from apparently normal tissue of the infected and the non-infected halves of the tubers was retarded to the same extent after addition of various inhibitors, namely with 90-95 % by 0.002 M. iodoacetate pH 6.2, with about 80 % by 0.02 M. sodium fluoride pH 6.2 and with 85-90 % by 0.002 M. sodium azide pH 6.2. In samples with a large increase of respiration after infection the inhibition by 0.06 M. malonate pH 4.3 proved to be lower (20-40 %) than in the other samples (65-75 %). Probably malonate did not attack succinic dehydrogenase exclusively. When a zone with soft tissue developed after infection, the softening of the tissue was accompanied by a decrease of the respiration rate and its inhibition by iodo- acetate and sodium fluoride. Data on the effect of the other inhibitors on the respiration of this tissue are not available. For the apparently normal tissue without mycelium it was concluded that the experiments with malonate were the only ones which indicated a qualitative change of the respiratory pathway; this effect however, was noticed only after continued influence of the infection. In the zone with soft tissue changes in the pathway had developed which were not observed outside this area. IV In the infected tuber halves starch disappeared from the tissue adjacent to the area with mycelium. In this tissue the fructose content did not change. Occasionally a more or less parallel behaviour of the respiration and of the amounts of sucrose and/or glucose was observed. The changes in the sugar content after infection were considered to be a parallel phenomenon resulting from the general activation of the metabolism, which also caused the increase of the 02-uptake, rather than the regulating mechanism of the respiration rate. V As the 02-uptake of healthy potato-tuber tissue was markedly stimulated by 10'3 M. 2,4-dinitrophenol (DNP) pH 5.0, the respiration rate was supposed to be regulated by the coupled phosphorylation, which in its turn depends on the activity of the transphosphorylating systems and the regeneration of phosphate-acceptor sites. The stimulation by DNP of the 02-uptake of apparently normal tissue from infected tuber halves decreased with the increase of the respiration after infection, which suggested a decrease of the rate-limiting action of the phosphorylation. Only in the tissue cut from the border between soft and apparently normal tissue the 02-uptake was not increased by DNP, indicating that the respiration was no longer limited by the phosphorylation. Although, especially in later stages of respiratory increase after infection, a qualitative change of the respiratory pathway could not be excluded, the hypothesis was put forward that in the apparently normal tissue of the infected tuber halves the regeneration of phosphate-acceptor sites was accelerated under the influence of the infection, in consequence of which the respiration rate could increase. Probably also the intensity and/or capacity of other processes which may become rate-limiting after acceleration of the phosphorylation increased with continued influence of the infection. VI Determinations of the total phosphate content showed that the fungus withdrew phosphorus-containing compounds from the non-invaded cells of soft tissue, not, however, from apparently normal tissue. In infected tuber halves without a zone with soft tissue a decrease of the inorganic phosphate and an increase of the organic phosphate was observed when respiration was markedly accelerated. In the apparently normal tissue of infected halves with soft tissue these two fractions were unchanged. In all infected halves the easily hydrolysable phosphate and its norit-adsorbed fraction tended to decrease with increasing respiration. Both fractions distinctly decreased in tissue cut from the border between soft and apparently normal tissue, which was probably a result of the withdrawal of phosphate-compounds by the fungus. The increase of organic phosphate after infection suggested an enhanced synthetic activity in the host cells, which may cause an accelerated phosphate-acceptor regeneration. The absence of this increase of organic phosphate in tuber halves with a zone with soft tissue is not necessarily in conflict with this view. VII The influence of the infection on the esterification of inorganic phosphate was investigated using P32-labelled phosphate (Na2HP3204). After the P32 taken up during 1.5 hours had been extracted with ethanol and water, the compounds n the extracts were separated by paper-chromatography. The distribution of the radioactivity on the paperstrip was determined. The radiation of the “nucleotides” (Rp < 1, except Rp 0.54), inorganic phosphate (Rp 1.0), “hexosephosphates” (Rp 1.10-1.50) and the compounds with Rp > 1.50 was calculated in % of the total radioactivity of the paperstrips. The % P32 in the “nucleotides” and the “hexosephosphates” appeared to increase, resp. decrease after infection. The influence exercised on the interpretation of these differences by a decrease after infection of the inorganic phosphate content in the host cells (Chapter vi), and by the fact that the P32-fraction which is insoluble in ethanol-water, was not included in the calculation of the %, was discussed. It was concluded that the increase of the % P32 in the “nucleotides” is an indication for the increase of the amount and/or the rate of conversion of the “nucleotides” and/or of the compounds of the insoluble P32-fraction. The decrease of the % P32 in the “hexosephosphates” does not necessarily mean that the synthesis and the rate of conversion of these compounds were retarded after infection. The results of the experiments with P32-labelled phosphate were considered to support the hypothesis that after infection the regeneration of phosphate-acceptor sites was accelerated, in consequence of which the respiration was increased. VIII The role which the wound-reaction that occurred after the tubers were cut into two halves, may have played in the respiratory increase in infected tuber halves, was discussed. Although the respiratory increase in the infected halves was most likely a result of the infection, a wound-reaction caused by the continual wounding of cells by the penetrating mycelium, might have played a From the results of the Chapters v, vi and vn the conclusion was drawn that it was not likely that in the apparently normal tissue of infected tuber halves an uncoupling of respiration and phosphorylation occurred. At least in the earlier stages of respiratory increase no evidence was obtained for a qualitative change of the respiratory pathway. In the soft tissue which developed in some infected tuber halves more changes were observed than in the apparently normal tissue. This might be connected with the dying of the cells.