Drug resistance and morphological variation in Phytophthora cactorum (Leb. and Cohn.) Schroet

A genetical study has been made of the homothallic phycomycete fungus Phytophthora cactorum. In the first section of the thesis techniques were developed for the production of asexual and sexual progeny from zoospores and oospores respectively. The survival of zoospores was found to be greatly improved if they were suspended in a solution containing 2% sucrose and Ringer's physiological salts rather than in water. The persistent dormancy of the oospores, which has thwarted genetical work with these fungi, was broken by passing them through the gut of the water snail Planorbis sp. Digested spores were surface sterillied in mercuric chloride, plated on non-nutrient agar and incubated four days in the light. By this means 66% germination of 25 day old oospores was obtained. In the absence of light germination was only 12% Section II deals with the search for drug resistant mutants. Eighteen common antibiotics, antimetabolites and other toxic substances were screened for their ability to inhibit mycelial growth and zoospore germination. Eight strains with increased tolerance to streptomycin were isolated when zoospores of the wild-type were incubated in drugged liquid medium. Some were resistant to 1000mug/ml streptomycin, i.e. ten times that tolerated by wild-type. One strain had an absolute requirement for streptomycin. The drug tolerance of the asexual and sexual progeny (self-fertilised) of one resistant strain, Sr, and the dependent strain, Sd, was examined. The resistance or dependence was inherited in each case unchanged and there was no evidence of segregation of individuals with a differing response. Absence of segregation could be taken to indicate either that the somatic nuclei are haploid or that resistance and dependence are determined extrachromosomally. Evidence was obtained which indicated that the presence of large numbers of streptomycin sensitive zoospores in drugged media inhibited the growth of added zoospores of the Sr strains. Non-dependent but resistant growths arose in the Sd strain when it was cultured on drug-free medium. Cultures with increased tolerance to sulphanilamide and acti-dione were obtained but resistance was found to be temporary and disappeared after growth in drug-free medium. Attempts to Induce stable drug resistance with ultraviolet irradiation were not successful. In Section III changes in colony morphology and their inheritance are described. Variation in colony morphology was found to occur sopntaneously and could also be induced with a high frequency on irradiation of zoospore with ultraviolet light. Treatment of zoospores for short periods with streptomycin induced unstable changes in morphology; reversion to wild-type always occurred in sub-cultures. An interesting pattern of somatic segregation of morphological characters was found which appeared to be determined extrachromosomally although proof of this will be obtained only when the observed segregation is compared with the behaviour of known nuclear markers. This variation first occurred in the streptomycin dependent strain but continued on its reversion to streptomycin independence. Colonies with wild-type morphology, derived from single uni-nucleate zoospores, gave rise to asexual progeny containing a proportion of fast growing, diffuse segregate. Wild-type colonies reappeared in asexual progeny of these segregants with low frequency. Single zoospores of the segregating line could be classified into two main groups according to the phenotype present in their asexual progeny. One interpretation suggested was that the zoospore contains a single determinant which exists in either of two forms and which mutates frequently from one form to the other. The proportion of the two phenotypes in the asexual progeny was found to change with ageing of the culture. The sexual progenies of these variants segregated in a similar way to the asexual progenies. So that large numbers of asexual progeny can be produced. Easily characterised nuclear markers are also required. Section I of this thesis deals with the development of cultural techniques which allow reproduction to be controlled. Section II is devoted to methods used in the isolation of nuclear markers and to an analysis of the inheritance of the mutant phenotypes. Section III deals with the patterns of inheritance shown by certain morphological variants