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ItemSexual reproductive structures of phytophthora vignae(University of Melbourne, 1992)Phytophthora vignae (Purss), an Oomycete, is a homothallic and amphigynous pathogen of cowpea, Vigna unguiculata (L.) Walp. In the sexual interaction in amphigynous Phytophthora the oogonial initial grows towards, contacts and penetrates the antheridial initial, subsequently maturation of gametangia, meiosis and fertilisation occur. In this thesis I have examined the surfaces of antheridia and oogonia of four races of P.vignae during development from reproductive initials to mature sexual structures, using fluorescein isothiocyanate (FITC)-labelled lectins, and a number of histochemical stains that react with carbohydrates. Changes in the nature and distribution of surface saccharide residues during maturation of sexual structures were observed. Cationic dyes containing basic collators bound most strongly to the walls of mature oogonia, indicating an increase in polyanions, possibly carboxyl-containing macromolecules. Lipid content also increased, as indicated by the development of Nile Red fluorescence under UV light. The lectins concanavalin A and Lens culinaris agglutinin bound strongly to developing and mature oogonial surfaces, indicating a high concentration of glucosyl/mannosyl residues on these surfaces. Peanut agglutinin bound strongly to developing reproductive structures, particularly presumptive antheridial initials, and It IS possible that an extended binding site for this lectin is present on the surfaces of these structures. Griffonia simplicifolia lectin bound to immature antheridia, suggesting the presence of N-acetyl glucosamine residues. Calcofluor White M2R New bound much more strongly to immature antheridia than to immature oogonia, indicating that difference in wall composition occurs early in development of reproductive secures. The difference in binding affinity continued to maturity. Scanning electron microscopy of developing reproductive structures has been used to provide further information about the surfaces involved and the morphology of -he reproductive structures: Die surfaces of antheridial and oogonial initials are similar in appearance to those of vegetative hyphae. However, mature oogonia have a distinctive granular surface. Scanning election microscopy of mature oogonial surfaces of other species of Phytophthora have shown that differences in surface ornamentation exist between species, so observations of the surface of mature oogonia of P.vignae may have some taxonomic value. Although P vignae is coenocytic, septa are formed at various times in the life cycle, for example, septa divide antheridia and oogonia from the parent hyphae, and septa or wall thickenings also are produced in old hyphae apparently separating dead or damaged portions from healthy sections. lectin binding studies have shown that the composition of these septa differs from that of hyphal walls, in that wheat germ agglutinin and Griffonia simplicifolia 11 lectin bind to hyphal septa, but not to hyphal walls, indicating the presence of oligomers of N-acetyl glucosamine in hyphal septa. The purified flurochrome from aniline blue, sirofluor, also bound more strongly to hyphal septa than to walls. Nuclear distribution during formation and maturation of sexual structures has been monitored using the DNA specific fluorochromes DAPI (4'6 diamidino-2-phenyl indole) and Hoechst 33258. The general pattern of nuclear behaviour is similar to that recorded for P.palmivora (Eriksson and Laane 1982) and P.erythroseptica, (Murphy, 1918) however the number of nuclei that enter the oogonium during the expansion phase is considerably less than that reported for other species of Phytophthora. The number of nuclei undergoing meiosis in the oogonium also differs. A comparative determination of die DNA content of nuclei of four races of P.vignae with an A2 isolate of P.cinnamomi and a haploid strain of Aspergillus nidulans has been made. The four races of P.vignae have similar nuclear DNA contents, indicating they are all of the same ploidy. The mean DNA content of P.cinnamomi nuclei has been found to be 1.6 times higher than that of P.vignae Race 1, and 5.2 times the mean DNA content of the haploid strain of A.nidulans tested. Appendix 3 gives the results of haemagglutination assays made on crude saline extracts of seeds of a range of species of Australian native plants. Lectins have been found in the seeds of a large number of plant species, so positive haemagglutination assays indicate the possibility that lectins may be present. Crude extracts of seeds of 34 species of indigenous Australian plants were tested for haemagglutination activity. Fourteen of the extracts agglutinated non-trypsinised erythrocytes to some extent, and two agglutinated trypsinised red blood cells only. Further tests indicated however that haemagglutination of two seed extracts selected for further study was probably due to phenolic substances.