Stimulation of sexual reproduction in Phytophthora cactorum by phospholipids is not due to sterol contamination

Abstract

Phytophthora cactorum did not form oospores on basal medium unless phosphatidylcholine (lecithin) or phosphatidylethanolamine (cephalin) was added. After removal of putative sterols by aminopropyl column chromatography, the activities of lecithin and cephalin were increased 47- and 2.8-fold, respectively, thus confirming the previous reports that sterols are not essential for sexual reproduction in this organism. Thin-layer chromatography (TLC) of the commercial lecithin revealed the presence of an unknown inhibitory substance which, when added to the purified lecithin, caused a 50% reduction of oospore formation. Commercial cephalin also showed a twofold increase in activity after removal of putative sterols and the existence of an unknown inhibitor when it was subjected to TLC. Addition of the inhibitor to the purified cephalin completely inhibited the growth of the test organism. One sample of lecithin tested was not stimulatory to oospore formation. However, after washing with deionized water or NaCl solution, it induced the production of 17300 and 24450 oospores (100 μg)-1, respectively. The ability of cephalin to induce oospore formation was increased 2⋅3-fold by washing with deionized water and 8⋅3-fold by washing with NaCl solution. Like sterols, the digitonin precipitable component (digitonide) of the non-phospholipid fraction of commercial lecithin or cephalin was stimulatory to oospore formation of P. cactorum but not Phytophthora parasitica. However, the non-digitonide component was not only more active than the digitonide component, but also stimulatory to P. parasitica. Gas chromatography and mass spectrometry (GC-MS) analysis of the digitonide component from lecithin failed to detect any putative sterol contaminant. The amount of the putative sterol contaminant in the digitonide component from cephalin was also below the detection limit of GC-MS. When 0.01-10 ng cholesterol was added to basal medium discs each containing 100 fig cephalin, the numbers of oospores produced by P. cactorum and P. parasitica were not significantly changed. It is concluded that, in the fungi tested, sterols did not play any significant role in the stimulation of sexual reproduction by highly purified phospholipids.