The nature of biotrophy in Phytophthora infestans

Phytophthora infestans, being an intermediate type between the facultative and obligate parasites among the Peronosporales (class Oomycetes ), was used to study the factors which are responsible for the inability of obligate parasites to grow axenically. In a comparative study on mitochondrial electron transport of Ph. infestans, Ph. erythroseptica and Pythium debaryanum, three species representing decreasing levels of specialization within the Peronosporales, the exceptional position of Ph. infestans was emphasized (papers I, II and III). The anomalous electron transport of the fungus is possibly the primary cause of general metabolic disorders, resulting in its complex nutritional requirements as described in paper IV.A method for the isolation of mitochondria from mycelium of P. debaryanum and Ph. infestans was developed. With this method, the structural integrity of the organelles of both species was essentially preserved; however, oxidative phosphorylation was partially uncoupled, indicating that mitochondrial function was not fully intact.Results from studies on respiration and cytochrome c reduction, using various substrates and specific inhibitors, indicate that in Ph. infestans as well as in Ph. erythroseptica and P. debaryanum mitochondria, an electron transport system similar to that described for other organisms was present. Cyanide-resistant respiration, as described for some higher plants, algae and fungi, was absent in these species.In Ph. infestans mitochondria respiration rates with the substrates pyruvate plus malate, α-oxoglutarate, and especially succinate, were much lower than in Ph. erythroseptica and P. debaryanum. Evidence was presented that a narrow-pass, located in the cytochrome(s) b region, limited the electron flux in the electron transport chain of Ph. infestans. Addition of the artificial electron transport mediator N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD) to the reaction mixture enhanced respiration by about 140% in the case of Ph. infestans, but only by 30% with P. debaryanum.In Ph. infestans an additional pathway was found to operate which permitted the oxidation of externally added NADH at a much higher rate than that of pyruvate plus malate, α-oxoglutarate and succinate. Its demonstration with both cytochrome c and oxygen as electron acceptors, its insensitivity to rotenone and antimycin A, and its sensitivity to cyanide indicate that this pathway is of mitochondrial origin, and connected with the main electron transport chain in the cytochrome c region, bypassing the postulated narrowpass. This pathway was also found to be present in Ph. erythroseptica and P. debaryanum mitochondria, but it did not contribute much to the oxidation of NADH in these species.Respiration of Ph. infestans, Ph. erythroseptica and P.debaryanum mitochondria with succinate as substrate was stimulated by low concentrations of ADP. This stimulation was due to the coupling of oxidative phosphorylation to respiration. higher concentrations of the chemical were inhibitory. The extent of inhibition was about the same in the three species. By employing specific inhibitors of mitochondrial respiration and different combinations of substrates and electron acceptors, the influence of ADP on several segments of the electron transport chain of the three fungi was measured. In this way, the site in inhibition by ADP was localized in the cytochrome c region of the electron transport chain. Because of the relatively high concentrations of ADP that are necessary for a significant inhibition and the similarity of the inhibition pattern in the three fungi tested, this anomalous inhibition of respiration by ADP is not likely related to the inability of Ph. infestans to grow on basal nutrient media.Linear growth rates of four strains of Ph. infestans on a defined nutrient medium containing glucose, some mineral salts, thiamine and a mixture of 13 amino acids were initially lower than on pea-juice agar, but during the subsequent logarithmic phase they became approximately the same on both media. Addition of other nitrogen sources such as glutamine, acid-hydrolized peptone, or an hydrolyzed fraction from peas to the basal nutrient medium resulted only in limited growth of these strains.The initial growth rate of three strains of Ph. infestans was increased by addition of soya-bean phospholipids to the basal medium plus amino acids; with one strain it was even higher than on pea-juice agar.No auxotrophy for any amino acid could be observed for any of the three strains tested. Omitting certain amino acids from the medium hardly affected growth, while omitting others reduced growth to a large extent. These effects varied with different strains of the fungus. Growth rates were also dependent on the concentration ratio of the amino acids. The results suggest that it is not possible to devise a synthetic nutrient medium meeting all the requirements of every strain of Ph. infestans.The demand of Ph. infestans for several amino acids at a time which can to some extent be replaced by others, is likely due to a limited activity of some pathways of intermediary metabolism. From this point of view, the limited activity of the mitochondrial electron transport could be the primary cause of the fungus' inability to synthesize sufficient amounts of some amino acids, possibly also of other low-molecular-weight compounds such as phospholipids. Good growth of the fungus is only guaranteed when such substances are provided from the host in vivo or added to the nutrient medium in vitro.