Physiology of sexual reproduction in Phytophthora parasitica

The ideal conditions for hormone production using Millipore filter adsorption technique were exposing both sides of the filter directly to freshly inoculated 40% V-8 agar block for 4 days. Two-time extractions with 95% ethanol could extract 100% of both hormone {dollar}alpha{dollar}1 and {dollar}alpha{dollar}2 adsorbed on Millipore filter. Hormone {dollar}alpha{dollar}1 was less polar than hormone {dollar}alpha{dollar}2 as shown by the results of solvent partition, thin layer chromatography and column chromatography. Alpha hormones were isolated from 40% V-8 agar culture of Phytophthora parasitica by extraction with a mixture of chloroform and methanol (2:1) for hormone {dollar}alpha{dollar}1 and 95% ethanol for hormone {dollar}alpha{dollar}2. Activity of hormone {dollar}alpha{dollar}1 or {dollar}alpha{dollar}2 was detected in organic phase after extraction with 2 N KOH and 2 N HCl. Neither hormone {dollar}alpha{dollar}1 nor {dollar}alpha{dollar}2 precipitated in cold acetone. Most of the {dollar}alpha{dollar}1 activity and all of the {dollar}alpha{dollar}2 activity were eluted from amino-propyl column in the fraction containing monoglycerides. Digitonin failed to precipitate either of the two hormones. Acetylation inactivated hormone {dollar}alpha{dollar}2 but not {dollar}alpha{dollar}1. Co-distillation test showed that {dollar}alpha{dollar}1 and {dollar}alpha{dollar}2 were high boiling point compounds and heat stable. Both hormone {dollar}alpha{dollar}1 and {dollar}alpha{dollar}2 survived saponification and the activity was found in non-saponifiable fraction. Alpha hormones were also isolated from cultures established on a chemically defined basal medium amended with non-saponifiable substances of sesame oil. GC-MC showed that a long chain alcohol was present in active {dollar}alpha{dollar}2 sample. Purification by florisil column chromatography increase the specific activity of both hormone {dollar}alpha{dollar}1 and {dollar}alpha{dollar}2. Sensitivity of various physiological processes to elevated temperature and light were investigated to determine the mechanisms of inhibition of oospore production at 34 C. The production of {dollar}alpha{dollar} hormones and their receptors by A1 and A2 isolates of Phytophthora was inhibited. Alpha hormones after being produced were stable at high temperature. No oospores were produced when cultures were exposed to 34 C during hormone reception or oospore formation after hormone induction. Light was found to be inhibitory to hormone production and hormone reception but not to the production of receptors or oospore formation after hormone induction. Alpha hormones were not sensitive to light.