Call number: LD2668 .T4 1952 A4 / Master of Science

Nutritional mutants of Ustilago hordel were used to demonstrate that parasexual recombination occurs within the host plant (Hordeum vulgare) prior to the production of teliospores. The nutritional mutants were also used to show that resistance of the newly-germinated seedling of H. vulgare to U. hordei and of subsequently formed tillers to infection was not correlated, and was probably not controlled by the same gene or genes. The application of gibberellic acid to H. vulgare was found to cause an increase in the overall tiller height of healthy plants by increasing the elongation of Internodal regions 0-1, 1-2, 2-3, and 3-4. There was no increased elongation of internodal regions 4-5 and 5-6. The healthy tillers of diseased plants showed no Internodal elongation when gibberellic acid was applied. Diseased tillers, which are usually shorter than healthy tillers, were also unaffected by the presence of gibberellic acid. The injection of both mating types of U. hordei into the young developing spike of a normally resistant strain of H. vulgare resulted in the production of diseased spikes. This would indicate that blockage to normal infection occurs at the time of seedling penetration, at the level of tiller primordia development, or at the time of spike primordia development. / Science, Faculty of / Botany, Department of / Graduate

Bibliography: leaves 218-232. / 232 leaves : ill. (some col.) ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / This thesis investigates the resistance response of barley towards R. secalis by examining the in vitro interaction between various components of the apoplastic fluid and the fungus. / Thesis (Ph.D.)--Adelaide University, Dept. of Applied and Molecular Ecology, 2001

M.Sc. / Barley (Hordeum vulgare) production is a vital constituent of the South African economy. Many pathogens reside on barley, which lead to low quality and yield. One of the most prominent barley pathogens, Fusarium graminearum, is the causal agent of small grain scab. F. graminearum resistance to barley is regulated by multiple genes referred to as quantitative trait loci (QTL), which makes it difficult to breed for resistance in new cultivars. Each of these genes contributes to a specific defence area and collectively counteracts Fusarium infection and spread in the barley plant. The aim of this project was to isolate and identify induced genes after infection of three leave stage barley with F. graminearum. These genes were isolated through the use of Suppression subtractive hybridisation (SSH), cloned and then sequenced. From this data set three transcript derived fragments (TDFs) sharing homology to known genes were selected and their expression profiles were studied through Northern blot analysis. Three TDFs shared homology with known genes namely a putative protease inhibitor-related protein, a senescence associated gene, and a manganese superoxide dismutase (MnSOD). These TDFs were previously also recognised for their function in host pathogen interactions. The expression analysis done using Northern blots showed up-regulation of the three fragments after inoculation. These results indicated that all the TDFs studied may play a role in the defence reaction of barley infected with F. graminearum, where both senescence and proteinase inhibitors could limit infection as well as spread and MnSODs might be a protective enzyme against oxidative stress. The results of this study indicated that all of the identified TDFs had database matches to proteins identified during stress responses. Furthermore, the Northern blot results indicated that all the TDFs studied could play a role in the defence reaction of F. graminearum infected barley. These TDFs will form the basis of further studies into the interaction between barley and F. graminearum. The results form this study will add to our knowledge of the interaction between barley and a necrotrophic pathogen. This will aid in understanding how cereal pathogens deal with pathogen attack and will aid in development of new more tolerant barley cultivars.

Genetic studies were carried out on the fungal parasite Ustilago hordei (Pers.) Lagerh. and on its host, Hordeum vulgave L. (cultivated barley). In these studies of the host-parasite relationship, special emphasis was placed on the genetic investigation of the pathogenicity. The thesis is divided into four parts. Part I deals with multial1 elism of genes for virulence (v-genes) in the parasite, and demonstrated that four different levels of virulence (obtained on the barley cultivar Trebi) are controlled by alternative alleles at a single genetic locus in the parasite. This is the first demonstrated example of a series of multiple alleles determining different levels of virulence. Part II concerns the identification and characterization of v-genes in U. hordei and of resistance genes (R-genes) in H. vulgave. Three v-genes (two of them new, one of them previously known) were identified. It was shown that the previously identified gene was expressed either as a dominant or a recessive, dependingoon the conditions under which it was tested, and that the newly-identified genes were both recessive. Cultures possessing the newly-discovered v-genes were used in identifying two new R-genes in the barley host. A study of interactions involving the newly discovered v- and R-genes led to the conclusion that these interactions have their basis in gene-for-gene relationships. Part III deals with the synthesis of a complex biotype of u. hordei possessing v-genes at two genetic loci. Disease reaction obtained with this complex biotype were compared both qualitatively and quantitatively with those obtained with the simpler, parental biotypes. In tests on certain cultivars the complex biotype produced either the same or higher levels of disease reaction. Because the new biotype has ah extended host range it is considered that under certain conditions it would be comparatively more fit than either of the parental biotypes from which it was derived. Part IV of the thesis concerns the effects of nutritional deficiency on the action of v-genes. Dikaryons which were homozygous for arg, ad or met were in all cases non-pathogenic; for those which were homozygous for pdx, pathogenicity was unaffected. For dikaryons which were heterozygous for one or more nutritional deficiences, pathogenicity was either unimpaired or reduced, depending on the combination (deficiency: virulence gene: host cultivar) which was tested. It was concluded that the specificity of pathogen biotypes was not determined by the availability or non-availability of specific nutritional factors. However, the effects were not entirely non-specific, since changes in levels of virulence were shown only in certain tests. / Science, Faculty of / Botany, Department of / Graduate

Call number: LD2668 .T4 1948 S34 / Master of Science

Bibliography: leaves 96-112 / iv, 156 leaves : ill ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.)--University of Adelaide, Dept. of Agronomy, Waite Agricultural Research Institute, 1986

The objective of this investigation was to assign the genes for resistance to leaf rust Puccinia hordei Otth. race 4 in two varieties of barley to the correct chromosome pair by means of crossing with nine translocation stocks. The varieties Sudan (C.I.6489) and Reka 1 (C.I.5051) were crossed with the following translocation stocks: 5051-2 (a-b), 5059-5 (a-e), 5030-2 (b-d), 5034-2 (b-f), 5062-3 (b-g), 5038-2 (c-d), 5056-2 (c-e), 5053-6 (e-f), and 5057-1 (f-g). The F₁ plants from crosses of Sudan with all translocations and Reka with all translocations were tested for rust reaction and found to be resistant. Examination of the mature pollen showed that the F₁’s were partially sterile. In the crosses involving the variety Sudan with translocation stocks having interchanges of chromosomes a with e and c with e, the F₂ data indicated complete linkage. The common chromosome e must carry the gene for resistance. This is substantiated by crosses involving translocations of a with other chromosomes and c with others, which showed independent assortment. In the crosses involving the variety Reka with translocation stocks having interchanges of chromosomes b with f and e with f, the F₂ data indicate that in Reka, the gene for resistance is linked with chromosome f. This is substantiated by crosses involving translocations of b with other chromosomes and e with others, which showed independent assortment. The results obtained from the F₂ data of the crosses studied indicated that in the varieties Sudan and Reka reaction to leaf rust is conditioned by genes that are independent of each other. / Ph. D.

Pyrenophora teres, the causal agent of net blotch of barley (Hordeum vulgare L.), exists in two forms; P. teres f. teres and P. teres f. maculata. Both forms induce a combination of brown necrotic spots and extensive chlorosis in susceptible barley cultivars. Although a number of low molecular weight compounds (LMWCs) have been previously isolated from P. teres culture filtrates, they only induced certain components of symptoms. Fungal metabolites were extracted from culture filtrates of both forms of the pathogen and separated into low (<3kDa) and high molecular weight compounds (HMWCs, >10 kDa) with each fraction inducing a component of the net blotch symptoms in a barley leaf toxicity assay. Inactivation of both LMWCs (<1kDa) and HMWCs resulted in loss of activity confirming their potential role in symptom development. Low molecular weight compounds induced chlorosis and water soaking but not the brown necrotic spots or lesions usually seen during the infection of barley by P. teres. The high molecular weight compounds (>10 kDa) induced the brown necrotic spots or lesions with no chlorosis evident. Further characterisation of LMWCs showed that they are not host specific while HMWCs exhibited host specificity. LMWCs were purified and further analysed using high voltage paper electrophoresis, staining and mass spectrometry. Electrophoretic properties and staining of the LMWCs with ninhydrin indicated that both forms of P. teres produced similar LMWCs in the conditions grown. Each form produced eight ninhydrin-positive compounds with the samerelative mobilities. Each individual compound was shown to induce chlorosis in excised barley leaves. All compounds except the one isolated in this study appear to be derivatives of or are the previously described compounds; N-(2-amino-2carboxyethyl) aspartic acid (Toxin A), aspergillomarasmine A, anhydroaspergillomarasmine A and aspergillomarasmine B. The exception is a bioactive UV absorbing LMWC which appears to be a reductive conjugation of the α-keto acid of phenylalanine with Toxin A. The HMWCs (>10kDa) were proteinaceous since they were identifiable using Coomassie staining. Additionally, the loss of activity that occurred with incubation at 40, 60, and 80 °C for 30 and 60 min followed a pattern fairly typical for protein denaturation. Further, treatment with protease decreased their phytotoxicity in proportion to the amount of enzyme used. Enzyme and heat treatment of proteins extracted from each form showed that proteins of P. teres f. teres are more resistant to heat and enzyme treatment compared with those of P. teres f. maculata. This suggests the protein(s) involved in symptom induction by P. teres f. teres and P. teres f. maculata are different which contributes to the difference in the symptom expression during the interaction between the pathogens and barley. Proteinaceous metabolites extracted from P. teres f. teres and P. teres f. maculata ranged from 10 to 100 kDa. Fractions purified using gel filtration had biological activity when they contained eight proteins when extracted from P. teres f. maculata (90, 80, 75, 55, 48, 35, 14 and 12 kDa) and six proteins when extracted from P. teres f. teres (90, 80, 55, 48, 14 and 12 kDa). Additionally, intercellular washing fluids (IWF) extracted from barley plants inoculated with both forms of P. teres, contained proteins of the same size as those in the biologically active fractions extracted from culture filtrates of P. teres f. maculata (80, 14 and 12 kDa) and P. teres f. teres (80, 48 and 14 kDa). Automated MS/MS sequencing of the biologically active proteins showed no resemblance to the sequences or conserved domain information available in public databases and as a consequence, these proteins were considered as novel proteins for P. teres. However, exact short matches with fragments resulting from the 80, 48 and 14 kDa proteins, showed considerable homology with ATP-binding cassette (ABC) transporters and their components, cellulases, serine proteinases as well as some hypothetical proteins isolated from different fungal species. Reaction of six plant species including one susceptible barley cultivar (Sloop) and one resistant line (CI9214) to P. teres showed that partially purified proteins induce the symptoms selectively in barley cultivars where the proteinaceous metabolites only induced brown necrotic spot/lesions in barley with a greater response seen on the susceptible cultivar Sloop when compared to the resistant line CI9214. No symptoms were seen on other plant species employed in this study suggesting that the proteinaceous metabolites isolated in this study are host specific phytotoxins. This research has allowed the first isolation of proteinaceous host-specific toxins from P. teres as well as the identification of a UV-sensitive LMWC phytotoxin not previously described. Proteinaceous toxins induced brown necrotic spots/lesions specific to the host while the LMWCs induced chlorosis in a number of different plant species. This contributes significantly to the body of knowledge defining how symptoms are caused during the pathogenicity process in the interaction between P. teres and barley. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1297672 / Thesis (Ph.D.) -- University of Adelaide, School of Agriculture, Food and Wine, 2007

Bibliography: leaves 121-141.