Septoria leaf blotch of wheat is a major biotic factor limiting the grain yield. To determine the nature of inheritance involving selected genotypes, three resistant semidwarf spring wheat lines exhibiting durable global resistance and one susceptible cultivar were crossed in all possible combinations, excluding reciprocals. Parents, Fl, F2, and F3 generations were inoculated with one pathogenic strain of Septoria tritici and evaluated under field conditions. Data were collected on an individual plant basis. F2 and F3 frequency distributions were computed to determine the nature of inheritance. Combining ability analysis of the 4x4 diallel cross and narrow-sense heritability were employed to estimate the nature of gene action. Phenotypic correlations were obtained to examined the possible association between disease severity traits and their relationship with heading date and plant height. The continuous distribution of the F2 and F3 populations among crosses made it impossible to classify plants into discrete classes in crosses between resistant x susceptible genotypes. Mean values of the disease traits Septoria progress coefficient, Relative coefficient of infection, and Septoria severity of flag leaf among the segregating populations were similar to the midparent values. Transgressive segregation was also observed in the F2 and F3 suggesting that parents had different resistance genes. Additive gene effects were found to be the major component of variation although nonadditive gene action played an important role in the expression of all three disease traits. The resistant parents Bobwhite"S" and Kavkaz /K4500 L.A.4 were found to have the largest negative general combining ability effects for the disease traits suggesting that these parents would be the best source for resistance to Septoria leaf blotch. High general combining ability and high narrow sense heritability estimates in the F3 population, indicated that substantial progress for resistance to Septoria tritici would be effective selecting in this generation. Of the three disease measures it would appear that selection for the lowest percentage of Septoria infection on the flag leaf would provide the most progress in developing resistant cultivars. Moderate and low negative phenotypic correlations were found among generations for the disease traits with heading date and plant height. From the results of this study the selection of early maturing short stature progeny would be possible within the genetic materials employed in this study. / Graduation date: 1993

The fungal pathogen Bipolaris sorokiniana (teleomorph Cochliobolus sativus)causes the foliar disease spot blotch (SB) and the root disease common root rot (CRR). Spot blotch and CRR are serious disease constraints to barley production in warmer growing regions of the world, with estimated yield losses ranging from 30-70% from SB and 15-30% for CRR. Although chemical treatments may assist incontrolling spot blotch infections, the most effective and environmentally sound means of control for each disease is breeding for varieties with natural resistance. InAustralia, no commercially available varieties offer resistance to either SB or CRR. This study has sought to establish molecular markers that will be useful for selecting for resistance to each of these important fungal diseases.Barley cultivars derived from the breeding line NDB112 have provided durable SB resistance in the North Dakota region of the USA for over 40 years. The robustnessof this resistance had not been determined under Australian environmental conditions or with those B. sorokiniana pathotypes present within Australia. Toelucidate the genetics of resistance, two seedling and two field trials were conducted on an ND11231-12/VB9524 (ND/VB) doubled haploid (DH) population (180 lines).A molecular map of the ND/VB population was curated in order to provide a firm basis for mapping of resistance loci. Composite interval mapping revealed thatdifferent gene combinations are effective at different stages of plant development. Seedling resistance was found to be conditioned by a major locus on the short arm ofchromosome 7H and this region was validated in the related population ND11231-11/WI2875*17. A minor quantitative locus on chromosome 5HS was detected in one of the two seedling trials. However, this region requires further investigation to confirm its association to SB resistance in this population. Field resistance to SB in adult plants was found to be associated with two major quantitative trait loci (QTL)on chromosomes 7HS and 3HS; and a putative third minor QTL on chromosome 2HS. The 7H region is common between seedling and field resistance and is the most important locus for the expression of resistance at both stages of plant development. These findings largely concur with genetic studies of this trait in tworowed barley germplasm in North American environments.Common root rot is a difficult disease to phenotype for, and breeding programs will benefit from the identification of molecular markers linked to resistance. Data wasprovided from field trials of subsets of the population over four years. Using a novel approach combining the efficiency of bulked-segregant analysis with highthroughputDiversity Arrays Technology markers (BSA-DArT), CRR resistance was found to be conditioned by three putative QTL in an unmapped Delta/Lindwall population. QTL were identified on chromosomes 2HS, 4HS, and 7HS. To validatethe trait-linkage associations between the DArT markers and the CRR QTL,microsatellite (SSR) markers known to map to the regions identified by BSA-DArT were used. The 2H and 4H regions were validated using marker regression of the SSR markers in most seedling trials, whereas the 7H QTL, which is proximal to the location of the SB resistance QTL in the ND/VB population, was detected in only one seedling trial.The QTL identified in this study offer potential to combat the foliar and root diseases causes by this fungal pathogen. The chromosomal location of QTL for SB and CRR resistance have been found to differ in the ND/VB and D/L populations,which suggests that resistance to each disease is independently inherited. Further research is required to confirm the hypothesis that it is possible to combineresistance to both diseases into a single genotype. Such allelic combinations would provide elite germplasm that would benefit barley breeding programs world-wide.

Thesis (Ph.D.)--Murdoch University, 2006. / Thesis submitted to the Division of Science and Engineering. Includes bibliographical references (leaves 330-343).

Thesis (Ph.D)--Murdoch University, 2007. / Thesis submitted to the Divsion of Health Sciences. Includes bibliographical references (leaves 215-250).

Studies presented in this thesis, highlight the complexity and importance of Mycosphaerella leaf disease (MLD) on Eucalyptus spp., especially in South Africa. In Chapter I, a review of the literature dealing with Mycosphaerella and MLD of Eucalyptus spp. is presented. It is clear from this review that the disease is prevalent in most countries where Eucalyptus spp. are commercially grown, including Australia where they are native. The number of Mycosphaerella species known from Eucalyptus spp. is increasing and this suggests that their economic effect on commercial Eucalyptus forestry, will probably Increase. It will thus become important to effectively identifY species responsible for MLD. To do this, the existing complex taxonomy of this group of fungi, will undoubtedly prove to be an obstacle. However, DNA based identification methods are proving useful in identifying species and delimiting lineages within Mycosphaerella. Future commercial propagation of Eucalyptus spp. will need to seriously consider the use of hybrids resistant to infection by Mycosphaerella spp. Furthermore, there will be a serious need for effective quarantine measures to prevent the introduction of new, perhaps more pathogenic, Mycosphaerella spp. into areas where they do not already occur. Three species of Mycosphaerella, M. molleriana, M. M. nubilosa and M. juvenis have traditionally been regarded as the most important Mycosphaerella spp. in South Africa. At various times, each species has been considered to be the only pathogen causing MLD in the country. Results from Chapter 2 and Chapter 3 have shown that M. nubilosa is the main pathogen responsible for MLD, especially, on E. nitens in the KwaZulu-Natal province of South Africa. This is interesting as M. molleriana, which was originally thought to be the only species in South Africa, was not isolated. Moreover, the susceptibility of E. nitens to M. nubilosa appears to be high, resulting in severe defoliation. Considering that E. nitens is a popular species grown at higher altitudes of South Africa, the recognition of M. nubilosa is important. This fungus is well recognized in Australia as an important pathogen and comparisons of data from that country will be useful in the future. Several Mycosphaerella spp. have, in the past been found to occur within single stands of commercial Eucalyptus spp. As part of the research presented in Chapter 3, surveys conducted in South Africa showed that there are seven species of Mycosphaerella occurring in plantations. These include: M. ellipsoidea, M. irregulariramosa, M. juvenis, M. lateralis, M. marksii, M. nubilosa and one newly described species M. fori. All of these species, apart from M. fori, were previously known to occur in South Africa. It is interesting that M. juvenis, previously thought to be one of the main species causing MLD, was found only to occur in a low numbers. This suggests that species causing epidemics may change over time. The identification of M. fori from a previously well surveyed area was unexpected. This new species was dominant in Tzaneen and future surveys will be conducted to determine its distribution and host range within South Afiica. The identification of a new species also highlights the need for additional surveys in South Africa to identify new species and to recognize possible new introductions of exotic Mycosphaerella spp. The presence of M. ellipsoidea, M. irregulariramosa, M. lateralis and M. marksii in this survey was not unusual, as they were previously known in South Africa. However, they were found only to occur at low levels and, as such, do not seem to contribute significantly to outbreaks of MLD.Various taxonomic and DNA-based methods have been used for the identification of Mycosphaerella spp. However, some taxonomic characters are of little value at the species level. In Chapter 4, RFLP's were considered as an option to differentiate between species of Mycosphaerella on Eucalyptus. Results of this study showed that the restriction enzyme HaeIII may be used for RFLP identification of Mycosphaerella spp. From a total of twenty-one Mycosphaerella spp. tested, RFLP digestion with HaeIII could resolve six of these species to species level. However, other species formed groups that had similar restriction profiles. They could be further separated based on ascospore germination patterns. This study forms a foundation for future studies in which other enzymes may be used together with HaeIIi to elucidate groups of species. It is suggested though, that this technique be combined with existing methods such as ascospore germination patterns and anamorph associations to identify species of Mycosphaerella occurring on Eucalyptus spp. with confidence. This should negate the use of expensive sequencing techniques, which are currently necessary. In virtually every country where Eucalyptus is grown commercially, MLD is prevalent. However, the specific Mycosphaerella spp. in countries are generally not the same. In Chapter 5, I used DNA sequence data from the ITS region of the rDNA operon as well as morphological data to identify M. heimii from Brazil and Hawaii, U.S.A. This represents the first report of the species from these countries. M heimii was previously thought to occur only in Madagascar and Indonesia, where it is recognized as a primary pathogen of several Eucalyptus spp, including E. urophylla. This is one of the main Eucalyptusspp. in Brazil. It has thus been suggested that this species may have been introduced into these countries via infected seed lots. This highlights the need for effective quarantine measures within these and other South American countries to inhibit the further spread of this pathogen through South America. Copyright / Dissertation (MSc)--University of Pretoria, 2011. / Microbiology and Plant Pathology / Unrestricted

El trabajo incluido en esta tesis está encuadrado en un proyecto cuyo objetivo general es el desarrollo de un vector viral eficiente de expresión o silenciamiento de genes, basado en el virus del manchado foliar de los cítricos (Citrus leaf blotch virus, CLBV). Para desarrollar un vector viral a partir del genoma de CLBV era necesario disponer de un clon infeccioso del virus y de métodos eficientes de inoculación del mismo en plantas de cítricos. Se construyó un clon infeccioso de cDNA del genoma completo de CLBV bajo el promotor T7 del fago lambda y se pusieron a punto protocolos para el aislamiento de protoplastos de N. benthamiana, N. occidentalis y cidro Etrog. CLBV replicó en protoplastos de las tres especies transfectados con viriones purificados, aunque el nivel de replicación fue muy bajo y sólo se detectó a partir de 4-5 días post inoculación (dpi), que es el tiempo máximo de supervivencia de los protoplastos en nuestras condiciones de trabajo. La multiplicación del virus en protoplastos inoculados con transcritos de CLBV fue menor, detectándose sólo en algunos experimentos de transfección de protoplastos de N. benthamiana. Finalmente, la inoculación mecánica directa de plantas de cítricos o de N. benthamiana y N. occidentalis con transcritos de RNA del virus fue infructuosa, a pesar de que estos transcritos eran capaces de infectar protoplastos. Antes de modificar el clon infeccioso de CLBV para convertirlo en un vector viral eficiente, era necesario conocer la estrategia de expresión del genoma viral y caracterizar las secuencias implicadas en el reconocimiento y promoción de la síntesis de los RNAs subgenómicos (sgRNAs) para poder duplicar un promotor y expresar genes o fragmentos de genes mediante la formación de un nuevo sgRNA. Para mapear el promotor del CP-sgRNA de CLBV se construyeron varios mutantes a partir del clon IC-CLBV (clon infeccioso del genoma completo de CLBV bajo el promotor 35S de CaMV) mediante supresión de nucleótidos y mutagénesis dirigida. / Renovell Ferrer, MÁ. (2010). El virus del manchado foliar de los cítricos: caracterización del promotor del RNA subgenómico del gen de la proteína de la cápsida y del supresor del silenciamiento de RNA [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/7324 / Palancia

Upon sensing pathogens, plants initiating defense responses typically resulting in programmed cell death (PCD). PCD effectively subdues biotrophic pathogens but is hijacked by necrotrophs that colonize the resulting dead tissues. We showed that barley wall associated kinase (WAK) genes, underlying the rcs5 QTL, are manipulated by the necrotrophic fungal pathogen Bipolaris sorokiniana to cause spot blotch disease. The rcs5 genetic interval was delimited to ~0.23 cM, representing an ~234 kb genomic region containing four WAK genes, designated HvWak2, Sbs1, Sbs2, and HvWak5. Post-transcriptional gene silencing of Sbs1&2 in the susceptible barley cultivars Steptoe and Harrington resulted in resistance, suggesting a dominant susceptibility function. Sbs1&2 expression is undetectable in barley prior to pathogen challenge; however, specific upregulation of Sbs1&2 occurred in the susceptible lines post inoculation. Promotor sequence polymorphisms were identified in the allele analysis of Sbs1&2 from eight resistant and two susceptible barley lines, which supported the possible role of promotor regulation by virulent isolates contributing to susceptibility. Apoplastic wash fluids from virulent isolates induced Sbs1expression, suggesting regulation by an apoplastic-secreted effector. Thus, the Sbs1&2 genes are the first susceptibility/resistance genes that confer resistance against spot blotch, a disease that threatens barley and wheat production worldwide. The nec3 mutants of barley are hyper-susceptible to many necrotrophs and show distinctive cream to orange necrotic lesions that are induced by infection, representing aberrant PCD. The γ- irradiation induced necrotic mutant, nec3-γ1 (Bowman) was confirmed as a nec3 mutant by allelism tests. The F2 progeny of a cross of nec3 x Quest inoculated with B. sorokiniana segregated as a single recessive gene fitting a 3 WT: 1 mutant ratio. The homozygous F2 mutant progeny were genotyped with four SSR and 25 SNP markers at nec3 locus on chromosome 6H, a physical region spanning ~ 16.96 Mb containing 91 high and low confidence annotated genes. Exome capture sequencing of nec3 mutants failed to identify a candidate gene, however, RNAseq analysis identified two candidates in the nec3 region with >three-fold downregulation. We hypothesize that the underlying aberrant PCD mechanism in the nec3 barley mutant facilitates extreme susceptibility to multiple adapted fungal pathogens of barley.

Net blotch is one of the most devastating diseases of barley (<i>Hordeum vulgare</i> L.) and occurs in two distinct forms, net-type net blotch (NTNB) and spot-type net blotch (STNB), caused by the fungal pathogens <i>Pyrenophora teres</i> f. sp. <i>teres</i> Smedeg. and <i>P</i>. <i>teres</i> f. sp. <i>maculata</i> Smedeg., respectively. Several sources of resistance have been previously reported, however, few barley cultivars with high levels of resistance have been developed from these sources. Efficient utilization of available resistance sources is dependent upon successful characterization of genes governing resistance in each resistant parent. Five net blotch resistant parents and one susceptible parent were crossed to identify novel resistance genes, postulate gene number and mode of inheritance, and conduct linkage mapping of novel genes for net blotch resistance. Results indicate that the highly resistant spring barley lines CIho 2291 and CIho 5098, and the winter barley cultivar Nomini each have single dominant genes for NTNB resistance. Resistance to NTNB in CIho 5098 is controlled by the same dominant gene conferring resistance in Nomini. Resistance to NTNB in CIho 2291 is controlled by one dominant gene which putatively is the same gene conferring resistance in ND B112, but differs from the resistance genes carried by the other parents in this study. An F2 population of 238 individuals derived from a cross between Nomini and the susceptible parent "Hector", and an F2 population of 193 individuals derived from a cross between CIho 2291 and Hector were used to map the genes governing NTNB resistance in Nomini and CIho 2291. The dominant gene governing resistance in Nomini, temporarily designated <i>Rpt-Nomini</i>, was mapped to a 9.2 cM region near the centromere of barley chromosome 6H between the flanking microsatellite markers Bmag0344a (r2=0.70) and Bmag0103a (r2=0.90), which were 6.8 cM and 2.4 cM away from <i>Rpt-Nomini</i>, respectively. The dominant gene governing resistance in CIho 2291, temporarily designated <i>Rpt-CIho2291</i>, was mapped to the distal region of barley chromosome 6H between the flanking microsatellite markers Bmag0173 (r2=0.65) and Bmag0500 (r2=0.26), which were 9.9 cM and 24.4 cM from <i>Rpt-CIho2291</i>, respectively. Previous studies have reported genes governing net blotch resistance in this region; however, allelism tests have not been conducted to determine the relationship between these genes. Identification of the chromosomal location of <i>Rpt-Nomini</i> and <i>Rpt-CIho2291</i> will facilitate future efforts in pyramiding multiple independent genes for net blotch resistance. / Ph. D.

Zymoseptoria tritici is an ascomycete fungus that causes Septoria tritici leaf blotch disease, which is one of the most devastating diseases of wheat. The lack of molecular tools has withheld functional genomics and consequently has left extensive gaps in the knowledge of the biology of infection by Z. tritici. The current research was conducted to develop molecular tools in order to facilitate forward and reserves genetic screens in Z. tritici. These tools include an optimised genetic manipulation protocol, the Z. tritici strains that provide high frequency targeted genome manipulations, a strategy for gene overexpression and protein tagging, and regulatable promoters for controlled gene expression in Z. tritici. The regulatable promoters served to reveal that the Z. tritici β-(1,3)- glucan synthase (BGS1) gene encoded an essential protein, which regulated cell wall stress tolerance and was therefore, a potential drug target. In addition, these molecular tools revealed a virulence-associated role of the glyoxylate cycle in Z. tritici as inactivation of this pathway impeded pre-penetration morphogenesis, which was restored by exogenous glucose application. This result implied that Z. tritici engaged the glyoxylate cycle to produce energy though gluconeogenesis by channelling the by-products of lipolysis. This significance of the glyoxylate cycle during initiation of the bi-phasic infection cycle suggests that Z. tritici is not a hemibiotroph, but a necrotrophic pathogen with an extended asymptomatic phase of infection. Overall, the molecular tools developed in this study will facilitate large-scale functional genomic analyses to interrogate the biology of infection by Z. tritici. The resulting data will inform the development of durable control strategies to combat Z. tritici outbreaks.

Biochar is the solid, carbon-rich by-product obtained from pyrolysis. It offers the prospect of long-term carbon sequestration and soil conditioning with agronomic benefits, often referred to as the ‘biochar effect‘. These multiple direct or indirect changes in the soil plant interface have also been associated with the control of plant diseases by influencing the host’s systemic induced resistance. The biological impact of biochar on the phytopathology of a major cereal grain has not yet been investigated. The most damaging foliar disease of barley in the UK is Rhynchosporium leaf blotch caused by the hemibiotrophic fungal pathogen Rhynchosporium commune. The aim of this project was to evaluate biochar performance and effectiveness as a disease control agent in the barley – R. commune – pathosystem, and attempted to identify underlying mechanisms by which biochar may function in the interaction between barley and the causal pathogen. Therefore, a unique set of nine slow pyrolysis biochars were established along a 350 to 800°C pyrolysis temperature gradient, with eight of these made from pelleted softwoods and a single one made from Miscanthus straw. A comprehensive biochar quality assessment was undertaken and established that the biochars differed in their chemical composition, which largely depended on production parameters, predominantly temperature (P < 0.05). The analysis proposed that biochar 9, made from Miscanthus at 800°C, showed added value as a soil conditioner over softwood biochars, due to higher pH, mineral ash and macronutrient recoveries, which pointed towards a possible liming potential. Regardless of the feedstock, biochars pyrolysed above 600°C indicated potential use for carbon sequestration purposes, due to higher carbon stability. Short-term controlled bioassays showed significant restricted growth of R. commune mycelium on defined medium to direct (1.0% w/w) and indirect volatile exposure from certain biochars (P < 0.001). The findings suggested a synergistic effect of the softwood biochars acidic nature and presence of fungicidal compounds, with observed inhibition of 100% attributed to re-condensation of tarry vapours onto biochar surfaces during pyrolysis. Qualitative biochar volatile organic compound analysis was conducted and identified biocide active phenolic and organic acid compounds, similar to those commonly found in smoke, bio-oils or wood vinegars. These findings proposed possible application for mitigation of inoculum pressure in field-grown barley, but the toxic nature of volatiles raised concerns over risks to human and environmental health, as also evidenced by detrimental barley growth effects. Subsequent controlled in vivo and in planta experiments revealed significant (P < 0.05) symptomatic barley leaf blotch reduction effects of up to 100%, following 5% (w/w) application of biochars 4, 5, 8 and 9. Barley plants transcriptional changes in ISR-dependent LOX2 and SAR-dependent PR1-b expression in planta verified systemic induced resistance as mechanisms behind the significant disease suppression of barley plants grown in soil amended with biochar 5 and 8. Disease reduction and biochar mediated induced resistance was attributed to either low concentrations of phytotoxic compounds, a direct toxicity effect from fungicidal compounds or indirect promotion of beneficial microbes. The results provided evidence, that in the case of the studied pathosystem, there is potential for biochar with specific characteristics to be considered as a soil amendment, offering not only carbon sequestration, but also possible improved disease resistance.