Pests and pathogens represent a serious and continuing threat to potato and tomato production worldwide. In this thesis, I have developed a new NB-LRRs probe library accounting for the recent improved annotations of both potato and tomato (Jupe et al., 2013 and Andolfo et al., 2014). The probe library was successfully used to map a late blight resistance in the diploid potato population B3C1HP. Using bulked-segregant resistance gene enrichment and sequencing (RenSeq) analysis in this population, which segregated 1:1 for the phenotype, the resistance was mapped to the lower end of chromosome 9. Furthermore, I developed a novel diagnostic tool, dRenSeq, to screen existing germplasm collection for the presence or absence of known, already characterised disease resistance genes, to prioritise novel resistances for research and breeding. dRenSeq was applied successfully on a set of S. okadae accessions as a proof of concept. The tomato late blight resistance gene Rpi-Ph3 was another focal point in this work, and the use of RenSeq was envisaged to identify Rpi-Ph3. However, another team published the gene (Zhang et al., 2014) and efforts were redirected towards the development of PCR markers to aid marker-assisted selection in breeding programs and to identify the cognate avirulence gene, Avr-Ph3. In addition, the new probe library was assessed in silico to evaluate if it would have enabled the identification of Rpi-Ph3 and homologous sequences. The identification of Avr-Ph3 was established through a large effector screen in an association panel of tomato accessions, co-infiltrations with Rpi-Ph3 in the model Solanaceae plant Nicotiana benthamiana and pathogen assays. The effector screen required the prior establishment of a robust transient expression system in tomato.

Phytophthora medicaginis is an important soil-borne oomycete pathogen of lucerne (Medicago sativa) and chickpea (Cicer arietinum) within Australia and overseas. To understand the host/pathogen interaction, a pathosystem was developed using the model legume Medicago truncatula. Using the resources developed for genetics and molecular characterisation in this model plant, the aim of this research was to understand the interaction between M. truncatula and P. medicaginis, with a view to improving resistance to this important pathogen in related legumes. To observe and characterise the interaction between M. truncatula and P. medicaginis, a pathosystem was developed by first screening a germplasm collection of 99 M. truncatula accessions. This revealed a continuous distribution in disease phenotypes with variable extremes in natural resistance to P. medicaginis culture UQ5750, isolated originally from M. sativa. P. medicaginis zoospore inoculation of 1-2 week-old seedlings in glasshouse experiments proved to be a robust and repeatable method to consistently confirm the responses observed for six key M. truncatula accessions; SA8618 and SA8623 exhibit high natural resistance to this pathogen, accession A17 is moderately resistant, A20 is moderately susceptible and accessions Borung and SA30199 are susceptible. To characterise the genetic basis of resistance to P. medicaginis, two reciprocal F2 populations from cross pollinations between A17 and Borung and SA8618 and SA30199 were produced and then phenotyped for disease symptoms. Genetic segregation patterns indicated the involvement of a gene with a major effect in both reciprocal populations. In particular, a 3:1 segregation ratio for resistance in the F2 populations from cross pollinations between A17 and Borung indicated the possibility of a single dominant gene for moderate resistance. Further phenotyping of F3 families is required to verify this. A M. truncatula linkage map was constructed using 50 F2 individuals of the A17 X Borung population and 49 F2 individuals from the Borung X A17 population. The map, covering 519.3 cM, is comprised of 84 SSR markers with an average distance between markers of 8.7 cM. These are evenly spaced over 7 linkage groups, including a super linkage group conferred by a translocation event between LG4 and LG8 of accession A17. Quantitative trait locus (QTL) analysis confirmed there was a QTL with a major effect in the A17/Borung reciprocal populations. A significant QTL was determined by quantifying two symptoms of P. medicaginis infection - proportion of dead/chlorotic leaves and root fresh weight. The trait loci for both symptoms were located on the same linkage group within the same region, supporting the putative position of the QTL and the authenticity of its involvement in resistance to P. medicaginis. This QTL was located on LG6 and accounted for 69.5% of the observed variation in proportion of dead/chlorotic leaves or 38.1% of the variation in root fresh weight within the inoculated populations. The effect of this QTL on resistance to P. medicaginis translated into 27.5% less dead/chlorotic leaves or 0.86 g more root fresh weight. Other QTLs with minor effects that are potentially involved in the interaction are located elsewhere on LG6 and LG2. However, the marker density of the linkage map and the population size need to be increased to verify this. In parallel to this, an F7 recombinant inbred line (RIL) population of chickpea (BG212 X Jimbour), developed by breeders at the New South Wales Department of Primary Industries (NSW DPI), was also assessed for the genetic basis of resistance to P. medicaginis. Variance component analysis of phenotype scores for this intraspecific RIL population indicated that 57.15% of the differences in between-family and withinfamily variance could be attributed to a genetic component. However, gene-based markers developed in M. truncatula and established simple sequence repeat (SSR) markers of chickpea were not sufficiently polymorphic in size to produce a linkage map for further QTL analysis. An interspecific cross between C. arietinum and C. echinospermum (Howzat X ILWC246) was also performed by breeders at the NSW DPI to develop RILs. In the duration of this research these interspecific RILs were bred to generation F3 and phenotyping assessment had not been performed. However, marker screening of the parents revealed 122 size polymorphic chickpea SSR markers. A sufficient linkage map could be produced for QTL analysis once field assessment of this population is performed. Initial screening of the M. truncatula gene-based markers on the parents of this interspecific cross also revealed that 50% show a sequence-identified base pair difference. A chickpea linkage map incorporating these markers could be comparatively mapped with M. truncatula. Molecular investigations of the M. truncatula/P. medicaginis pathosystem were performed to elucidate the possible underlying defence mechanisms involved in the observed resistance. To determine the function of ethylene in the resistant response, the characterisation of defence associated mutants of M. truncatula and Agrobacterium rhizogenes-mediated ‘hairy root’ transformations were employed. Comparison of response to inoculation of an ethylene insensitive mutant of M. truncatula (sickle) with the moderately resistant background genotype A17 showed that sickle was hypersensitive to P. medicaginis. This indicated that ethylene insensitivity was not the source of resistance to this pathogen and importantly that ethylene is a key defence signalling molecule in the moderate resistance of A17 to P. medicaginis. Agrobacterium-mediated ‘hairy root’ transformations of M. truncatula with 4GCC::Luc constructs, revealed that the production of ethylene and consequently ethylene response factors (ERFs) after inoculation by P. medicaginis was a general defence reaction by all accessions. The two susceptible M. truncatula accessions exhibited a much stronger and earlier response to inoculation than the highly resistant and moderately resistant accessions. This indicated that the resistant response may be directed by a transcriptional component governed by the host genotype, downstream of ethylene production. The M. truncatula/P. medicaginis ‘hairy root’ transformation assay has scope to be a powerful functional genomics tool for this pathogen interaction. Reverse transcriptase quantitative polymerase chain reaction (RTqPCR) was employed to determine the general patterns of gene expression and function underlying the response to P. medicaginis infection. Relative changes in gene expression of key enzymes in each of the salicylic acid, jasmonic acid, ethylene and isoflavonoid defence pathways and in genes encoding downstream target proteins revealed potential genes involved in the resistance to P. medicaginis. There was a distinct molecular difference in the response between the high and moderately resistant M. truncatula phenotypes to this pathogen. Moderate resistance to P. medicaginis in M. truncatula is possibly mediated by ethylene and involves the considerable induction of pathogenesis related protein 5 (PR5), which was not the same defence response that conferred the high resistance to P. medicaginis. Early and consistent expression of genes encoding key enzymes of the isoflavonoid pathway by the highly resistant accession indicated that phytoalexin response could be associated with the high resistance. Confirmation of the involvement of isoflavonoid phytoalexins in the high resistance response to P. medicaginis merits further investigation.

Phosphite has been used to effectively control the soil borne plant pathogen Phytophthora cinnamomi in many horticultural crops, forest trees and natural ecosystems. However, the molecular mechanisms behind phosphite action on this pathogen are poorly understood. Several studies have shown that phosphite inhibits growth and zoospore production of P. cinnamomi and in addition induces significant physiological and metabolic changes in the mycelium. As an approach to understanding the mechanisms and relevance of these changes in the pathogen, the effect of phosphite on gene expression was investigated using microarray analysis. To construct the microarray, RNA was extracted from phosphite-treated (40 ug/ml) mycelium of P. cinnamomi isolate MP 80. The chosen phosphite concentration inhibited the mycelial growth by 70% but provided sufficient mycelium for RNA extractions after 4 days growth at 25C. The mRNA was reverse transcribed into cDNA and cloned into lambda to construct a library consisting of 2 million pfu of which 80 % were recombinant phage. The inserts were sequenced for a random selection of clones from the library. The nucleotide sequences generated revealed a range of different P. cinnamomi genes being expressed and demonstrated that the cDNA library provided a good representation of the transcripts expressed in P. cinnamomi. The types of genes found to be expressed in the mycelium of P. cinnamomi included genes encoding GTP binding proteins involved in vesicle transport, structural proteins involved in maintaining cell membrane integrity,elicitors, phosphatases and ribosomal proteins. Over nine thousand cDNA transcripts were randomly selected from the cDNA library and prepared by PCR amplification and purification for microarray construction. Custom made cDNA arrays containing 9216 cDNA transcripts were constructed and probed with RNA from untreated mycelium and mycelium grown in medium with 40 ug/ml phosphite. Two genes, EF-1 alpha and cinnamomin gene, identified by qRT-PCR as being constitutively expressed were also positioned on the arrays as positive controls. In the process of identifying constitutively expressed genes, qRT PCR revealed that phosphite down-regulated a gene encoding ubiquitin-conjugating enzyme, a component of the ubiquitin/proteasome pathway involved in the removal of abnormal and short lived-regulatory proteins and rate limiting enzymes. From the arrays a further seventy-two transcripts with altered patterns in gene expression (fold change > 2) were identified. The majority of the cDNA transcripts spotted on the array were down-regulated with changes in gene expression ranging from 2- to 3.5-fold. Thirty-two cDNA transcripts were up-regulated with changes in gene expression ranging from 2- to 16-fold. Characterisation by sequencing revealed that the most highly induced transcripts coded for ADP-ribosylation factors, an ABC cassette transporter and a glycosyl transferase. A transcript encoding a vitamin B6 biosynthesis protein was also identified as up-regulated by 2.9-fold. In contrast, the down-regulated transcripts coded for cellulose synthase I, annexin, glutamine synthetase, metallothionein and an alternative oxidase. The results are discussed in terms of possible roles and mechanism(s) of phosphite action within the mycelium of P.cinnamomi. This work is the first comprehensive screen for phosphite regulated-gene expression in P. cinnamomi and represents a significant step towards an understanding of the mode of action of phosphite on this organism. This thesis provides valuable information on the molecular interaction between phosphite and P. cinnamomi, which in future studies may stimulate the discovery of novel methods and cellular targets for the control of plant pathogenic Oomycetes.

One of the major aims of the research presented in this thesis was to assist managers of native vegetation communities in southeastern Australia in understanding the dynamics of P. cinnamomi with an important ecological species, Xanthorrhoea australis. It trialed the use of phosphite in large-scale field applications to establish the usefulness of this management option for the first time on Victorian flora. This thesis describes the process of disease development within mature X. Australia plants. For the first time it was shown that within X. australis plants, secondary disease symptoms are related to the percentage of stem that has been infested by the disease. It was evident that after initial invasion the pathogen moves via root xylem and throughout the plant within vascular to the stem, especially within the desmium. The research shows that the pathogen could not be isolated consistently even though it was considered to be responsible for disease symptoms. Trials of a control fungicide (Foli-R-fos 200) shows that protection occurs in many susceptible plants when 2 and 6g a.i./L phosphite is applied. Phytotoxicity occurred in native plants at Anglesea and within controlled environment trials when using ≥ 6g a.i./L. It will be shown that 2g a.i./L phosphite controls disease in sprayed plots within heathlands at Anglesea and a recently burnt coastal woodland community at Wilson’s Promontory. The proportion of healthy X. australis plants treated with phosphite was significantly higher than the proportion in control plots without phosphite. The research shows that phosphite was recovered from leaves of three species treated with Foli-R-fos 200 in the field. For the first time it has been shown that seed germination was reduced in two species when high concentrations of phosphite were applied. The first documentation of the effect that phosphite has on soil properties showed that nitrogen and oxidised organic carbon were the only parameters to alter significantly. This thesis provides answers to some important questions, answers that can now be used by managers in formulating better policies and actions at an operational level. There has been a dire need in Victoria to address many issues regarding P. cinnamomi and this thesis provides relevant and informative approaches to disease control, and a better understanding of the disease progress.

Bibliography: leaves 227-248.

In 1991 Pacific yew (Taxus brevifolia Nutt.) was reported as a new host for Phytophthora lateralis Tucker and Milbrath which is an aggressive root rot pathogen thought previously to be specific to Port-Orford-cedar. This study was designed to compare the pathogenicity of P. lateralis on the two hosts, and to characterize sites where Pacific yew mortality occurs. The specific objectives were: 1) compare root colonization and mortality of Pacific yew and Port-Orford-cedar seedlings and rooted cuttings; 2) compare lesion length on inoculated Pacific yew and Port-Orford-cedar branches and stems; 3) compare zoospore attraction to freshly cut Pacific yew and Port-Orford-cedar rootlets; 4) compare amount of mortality of Pacific yew and Port-Orford-cedar in infested drainages and determine extent of yew mortality; and 5) characterize sites where P. lateralis causes Pacific yew mortality. Root colonization of P. lateralis was significantly greater in cedar than in yew. Seedling mortality averaged 58% for cedar and 4% for yew. Lesion length on the cedar seedling stems was twice the lesion length on yew stems, and cedar branches had lesions four times longer than yew branches. Abundant zoospore aggregation occurred on cedar rootlets along the zone of elongation and the region of maturation. In comparison, far fewer zoospores encysted along the yew rootlets, and were concentrated on the root hairs. The stream survey of three infested drainages in southwest Oregon and northwest California revealed a total of 1199 dead Port-Orford-cedar (46% mortality), and 86 dead Pacific yew (10% mortality). At sites where P. lateralis-induced mortality occurred, the interaction of slope and distance from the stream was negatively correlated with tree death. Based on results of this study, we conclude that Pacific yew is less susceptible to P. lateralis than Port-Orford-cedar, but that mortality of Pacific yew in the field is greater than previously reported. In addition, Pacific yew mortality was observed most often on level to nearly-level sites close to the stream's edge where root exposure to P. lateralis-infested water was frequent in scope and duration. / Graduation date: 1995

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Typescript. / Thesis (Ph. D.)--University of Hawaii at Manoa, 1977. / Bibliography: leaves 150-154. / Microfiche. / xvi, 154 leaves ill

Bibliography: leaves 227-248. / xx, 248 leaves, [22] leaves of plates : ill. (chiefly col.) ; 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 Crop Protection, 1995