Tesis sobre el tema "Non-host plants"

Pest control strategies targeting insect olfaction represent a promising venue for control of tortricid insects (Lepidoptera: Tortricidae). Among tortricids, the grapevine moth Lobesia botrana (Denis and Schiffermüller) and the codling moth Cydia pomonella (L.) are serious pests for worldwide production of fruit crops. We employed several approaches to the olfactory system, from electrophysiological and behavioral studies in the grapevine moth, to bioinformatic and molecular studies of olfactory sensory proteins in the codling moth. At the receptor level, we studied both the Olfactory Receptors (ORs), the most common class of sensory proteins mediating detection of odors in insect antennae, and the Transient Receptor Potential (TRP) channels, a novel family of receptor, that recently were also found in the antennae of lepidopterous species. We demonstrated electrophysiological and behavioral responses of the grapevine moth to volatiles emitted by a non-host, Perilla frutescens, previously known to activate TRPs in the rat, Rattus norvegicus. In the codling moth, we characterized a novel TRP channel (TRPA pyrexia-like) and we confirmed activation of its human orthologue to the same non-host compounds active on the olfactory system of the grapevine moth. ORs were heterologously expressed in vivo and in vitro, for identification of their ligands among host and non-host plant volatiles and pheromones (deorphanization). Among several ORs of codling moth, we deorphanized a candidate pheromone receptor (PR) to plant synergists, an OR to non-host volatiles and another PR candidate to a pheromone antagonist of the insect. Our study thus opens for refinement of existing pest control, or novel applications. The behavioral response of the grapevine moth to volatiles from a nonhost plant, and the identification of a novel TRP channel in the codling moth may have perspectives for application in agriculture, targeting the somatosensory system of these tortricids. The evolutionary implications of the responses of the human orthologue of TRPA pyrexia-like to volatiles active on the grapevine moth olfactory system could imply a large degree of conservation of the receptor function. In the codling moth, identification of synergist and antagonist ligands for candidate PRs and deorphanization of an OR to non-host plant volatiles suggest a possible role of these receptors in reproductive and ecological isolation. This could lead to further refinement of existing semiochemicalbased control techniques, by enabling a better understanding of mate- and host-finding in this species.

Plant innate immunity is activated upon pathogen attack by recognizing their avirulent (avr) genes by Resistant (R) genes leading to R-gene resistance or host resistance. Another form of innate immunity is non-host resistance that is exhibited by a given plant species to most strains of a microbial species. R-gene resistance activates salicylic acid (SA) that is synthesized from methyl salicylic acid (MeSA) by Salicylic Acid Binding Protein 2 (SABP2). It was hypothesized that SABP2 plays the similar role in non-host resistance also. Growth experiments and non-host related gene analysis experiments were conducted on tobacco plants using P.s tabaci and P.s. phaseolicola that are host and non-host pathogens on tobacco respectively. Tobacco control plant C3 that expresses SABP2 and 1-2 that is RNAi silenced in SABP2 expression were used in this study. Results suggest that SABP2 may not have any significant role in tobacco non-host resistance.

Stem rust, caused by the macrocyclic fungal pathogen P. graminis (Pg), is one of the most devastating diseases of wheat and other small grains globally; and the emergence of new stem rust races virulent on deployed resistance genes brings urgency to the discovery of more durable sources of genetic resistance. Given its intrinsic durability and effectiveness across a broad range of pathogens, non-host resistance (NHR) presents a compelling strategy for achieving long-term rust control in wheat. However, NHR to Pg (Pg-NHR) remains largely unexplored as a protection strategy in wheat, in part due to the challenge of developing a genetically tractable system in which Pg-NHR segregates. In this dissertation, an investigation of Pg-NHR is undertaken via the pathogen's alternate (sexual) host, barberry ( Berberis spp.). Within the highly diverse Berberis genus, numerous species function as alternate hosts to Pg but others are non-hosts. European barberry (B. vulgaris L.), for example, is susceptible to Pg infection but Japanese barberry (B. thunbergii DC.) is a non-host. In this study, the nothospecies B. ×ottawensis C.K. Scheid, an inter-specific hybrid between Pg-susceptible B. vulgaris and Pg-resistant B. thunbergii, is explored as a possible means of mapping the gene(s) underlying the apparent Pg-NHR exhibited by B. thunbergii. The overall goal of this research is to contribute to the global search for novel sources of potentially durable stem rust resistance genes.

The first chapter describes a field study conducted in western Massachusetts, in which a natural population of B. ×ottawensis was characterized to determine if the hybrid can be used to genetically dissect the Pg-NHR exhibited by B. thunbergii. A population of 63 B. ×ottawensis individuals were clonally propagated, phenotyped for disease response to Pg via controlled inoculation using overwintered telia of Pg found on naturally infected E. repens, and genotyped using the de novo genotyping-by-sequencing (GBS) pipeline GBS-SNP-CROP. Controlled inoculation of a subset of 53 B. ×ottawensis accessions, verified via GBS to be true, first-generation hybrids, revealed 51% susceptible, 33% resistant, and 16% intermediate phenotypes. Although such variation in disease response within a natural population of F₁ hybrids could be explained by non-nuclear (cytoplasmic) inheritance of resistance, a similar pattern of segregation was observed in a population of B. ×ottawensis full-sibs, developed via controlled crosses. The results of this first chapter demonstrate not only that the Pg-NHR observed in B. thunbergii segregates among F₁ interspecific hybrids with Pg-susceptible B. vulgaris but that the resistance is likely nuclearly inherited. Therefore, at least in principle, the gene(s) underlying Pg-NHR in B. thunbergii should be mappable in an F₁ population derived from the controlled hybridization of the two parental species.

Building on the results of first chapter, the second chapter of this dissertation details the generation and use of a bi-parental B. ×ottawensis mapping population to develop genetic linkage maps for both parental species and begin mapping the gene(s) underlying Pg-NHR in B. thunbergii. Using 162 full-sib F₁ hybrids and a total of 15,411 sequence variants (SNPs and indels) identified between the parents via GBS, genetic linkage maps with 1,757 and 706 markers were constructed for B. thunbergii accession 'BtUCONN1' and B. vulgaris accession 'Wagon Hill', respectively. In each map, the markers segregated into 14 linkage groups, in agreement with the 14 chromosomes present in these Berberis spp. The total lengths of the linkage maps were 1474 cM (B. thunbergii) and 1714 cM (B. vulgaris), with average distances between markers of 2.6 cM and 5.5 cM. QTL analysis for Pg resistance led to the identification of a single QTL, dubbed QPgr-3S, on the short arm of chromosome 3 of B. thunbergii. The peak LOD score of QPgr-3S is 28.2, and the QTL spans 13 cM, bounded by the distal SNP marker M411 and proximal SNP marker M969. To gain further insight into the QPgr-3S region, a chromosome-level 1.2 Gb draft genome for B. thunbergii was assembled using long PacBio reads and Hi-C data. By anchoring the B. thunbergii linkage map to the draft genome, the 13 cM Q Pgr-3S region was found to correspond to ~3.4 Mbp, represented by 10 contigs. Using a 189.3 Mb transcriptome assembled from a multiple tissue library of RNA-seq data, the QPgr-3S region was found to contain 99 genes. To help narrow this list to candidate genes of highest priority for subsequent investigation, a combination of approaches was taken. Specifically, annotation of the QTL region and differential gene expression analysis led to the identification of 12 candidate genes within the region. (Abstract shortened by ProQuest.)

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A brusone, causada por Pyricularia oryzae, é considerada uma doença economicamente importante para trigo na América do Sul. Uma das estratégias de manejo para minimizar as perdas causadas por essa doença é o uso de cultivares resistentes. Alternativamente, pode-se utilizar indutores de resistência para aumentar o nível basal de resistência do trigo à brusone. O presente estudo teve como objetivos: i) determinar as alterações fisiológicas e bioquímicas em plantas de trigo pulverizadas com uma concentração não fitotóxica do ácido α-picolinico (AP), o qual é uma toxina não seletiva produzida por P. oryzae e ii) verificar a manipulação metabólica exercido por P. oryzae quando infectando cultivares de trigo com diferentes níveis de resistência basal à brusone. Nas folhas de trigo pulverizadas com uma concentração não fitotóxica de AP (0.1 mg mL -1 ), os sintomas da brusone desenvolveram menos em associação com um melhor desempenho fotossintético, melhoria do metabolismo antioxidante e redução nas concentrações de H 2 O 2 , O 2 ●- e MDA. As cultivares BR-18 e EMBRAPA- 16 foram mais resistentes à brusone do que a cultivar BRS-Guamirim. O desempenho fotossintético das plantas infectadas foi alterado devido a limitações de natureza difusiva e bioquímica para uma eficiente fixação do CO 2 . Durante a fase assintomática da infecção por P. oryzae, mudanças drásticas no metabolismo de carboidratos e nos níveis de aminoácidos, compostos intermediários do ciclo de Krebs e poliaminas ocorreram nas plantas das três cultivares sugerindo, assim, uma manipulação metabólica exercida por P. oryzae. No entanto, um metabolismo antioxidativo mais eficiente foi importante para neutralizar os efeitos deletérios da infecção por P. oryzae em associação com maiores atividades da fenilalanina amônia liase e polifenoloxidase e maiores concentrações de compostos fenólicos e lignina. Com base nesses resultados e possível concluir que a concentração não fitotóxica de AP foi capaz de potencializar a defesa das plantas de trigo e reduzir a severidade da brusone. A infecção do trigo por P. oryzae ocasionou distúrbios no metabolismo primário das plantas e alguns deles foram semelhantes entre as cultivares independentemente do nível basal de resistência delas.
Blast, caused by Pyricularia oryzae, has become an economically important disease in wheat in South America. One of the management strategies for minimizing the losses caused by blast includes the use of resistant cultivars. Alternatively, the use of inducers of resistance showed the potentiation to increase wheat resistance to blast. This study aimed: i) to determine the physiological and biochemical alterations in wheat plants sprayed with a non-phytotoxic concentration of α-picolinic acid (PA), which is a non-host selective toxin produced by P. oryzae and ii) to establish the degree of metabolic manipulation exerted during the infection by P. oryzae on plants from cultivars with different levels of basal resistance to blast. The spray of leaves of plants with a non-phytotoxic concentration of PA (0.1 mg mL -1 ) resulted in less blast symptoms in association with a better photosynthetic performance, an improvement on the antioxidant metabolism and reduced concentrations of H 2 O 2 , O 2 ●- and malondialdehyde. The cultivars BR-18 and EMBRAPA-16 were more resistant to blast in comparison to cultivar BRS-Guamirim. The photosynthetic performance of the infected plants was altered due to diffusional and biochemical limitations for CO 2 fixation. During the asymptomatic phase of P. oryzae infection, drastic changes in the carbohydrates metabolism and on the levels of amino acids, intermediates compounds of Krebs cycle and polyamines occurred on plants from the three cultivars suggesting a metabolic manipulation exerted by the pathogen. However, amore efficient antioxidant metabolism was able to help the wheat plants to counteract against the deleterious effects of P. oryzae infection in association with great phenylalanine ammonia lyases and polyphenoloxidases activities and high concentrations of phenolics and lignin. Based on this information, it is possible to conclude that a non- phytotoxic concentration of PA elicited the activation of host defense mechanisms that reduced blast severity. Likewise, the infection of leaves by P. oryzae induced remarkable disturbances in the primary metabolism and some of them were conserved among the cultivars regardless of their basal level of resistance to blast.

Dans cette thèse, j'ai étudié le rôle des sRNAs dérivés de l'hôte et du virus lors de l'infection du colza (Brassica napus, Canola) par la souche UK1 du virus de la mosaïque du navet (TuMV-UK1). En utilisant un dérivé de TuMV fusionné avec un gène codant pour la protéine fluorescente verte (TuMV-GFP), deux cultivars de colza (‘Drakkar’ et ‘Tanto’) qui diffèrent par leur susceptibilité à ce virus ont été identifiés. Le profil transcriptionnel des foyers d'infection locale, dans les feuilles de Drakkar et de Tanto, par séquençage nouvelle génération (NGS) a révélé de nombreux gènes exprimés de manière différentielle. Les mêmes échantillons d'ARN provenant de feuilles de Drakkar et de Tanto, traitées par des virus ou utilisées en contrôle, ont également servi à établir le profil NGS des sRNAs (sRNAseq) et de leurs cibles potentielles d'ARN (PAREseq). Les analyses bioinformatiques et leur validation in vivo, ont permis d’identifier les événements de clivage de transcrits impliquant des micro ARN (miRNA) connus et encore inconnus. Fait important, les résultats indiquent que TuMV détourne la voie du RNA silencing de l’hôte avec des siRNAs issus de son propre génome (vsiRNA) pour cibler les gènes de l’hôtes. Le virus déclenche également le ciblage à grande échelle des ARN messagers (ARNm) de l’hôte par l’activation de la production de siRNAs secondaires en phase, à partir de locus PHAS. À leur tour, les vsiRNAs et les siRNAs dérivés de l'hôte (hsRNAs) ciblent et clivent l'ARN viral par le complexe RISC. Ces observations éclairent le rôle des siRNAs dérivés de l'hôte et du virus dans la coordination de l'infection virale. Un autre chapitre de cette thèse est consacré à l'analyse des maladies induites par des virus en utilisant comme modèle de plante Arabidopsis, infectée par un tobamovirus, le virus de la mosaïque du colza (ORMV). De plus, ces observations ont permis de proposer un modèle dans lequel cette guérison dépend d’un adressage important de vsiRNAs secondaires antiviraux depuis leur source de production jusqu’à leurs tissus de destination, et l'établissement d'un apport en vsiRNAs capable de bloquer l'activité VSR impliquée dans la formation des feuilles symptomatiques
In this thesis, I investigated the role of host- and virus-derived sRNAs during infection of Rapeseed (Brassica napus, Canola) by the UK1 strain of Turnip mosaic virus (TuMV-UK1). By using a TuMV derivative tagged with a gene encoding green fluorescent protein (TuMV-GFP), two rapeseed cultivars (‘Drakkar’ and ‘Tanto’) that differ in susceptibility to this virus were identified. Transcriptional profiling of local infection foci in Drakkar and Tanto leaves by next generation sequencing (NGS) revealed numerous differentially expressed genes. The same RNA samples from mock- and virus- treated Drakkar and Tanto leaves were also used for the global NGS profiling of sRNAs (sRNAseq) and their potential RNA targets (PAREseq). The bioinformatic analysis and their in vivo validation led to the identification of transcript cleavage events involving known and yet unknown miRNAs. Importantly, the results indicate that TuMV hijacks the host RNA silencing pathway with siRNAs derived from its own genome (vsiRNAs) to target host genes. The virus also triggers the widespread targeting of host messenger RNAs (mRNAs) through activation of phased, secondary siRNA production from PHAS loci. In turn, both vsiRNAs and host-derived siRNAs (hsRNAs) target and cleave the viral RNA by the RISC-mediated pathway. These observations illuminate the role of host and virus-derived sRNAs in the coordination of virus infection. Another chapter of this thesis is dedicated to the analysis of virus-induced diseases by using Arabidopsis plants infected with the Oilseed rape mosaic tobamovirus (ORMV) as a model. Initially, the infected plants develop leaves with strong disease symptoms. However, at a later stage, disease-free, “recovered” leaves start to appear. Analysis of symptoms recovery led to the identification of a mechanism in which the VSR and virus derived-siRNAs play a central role. I used Arabidopsis mutants impaired in transcriptional and post-transcriptional silencing pathways (TGS and PTGS respectively) and a plant line carrying a promoter-driven GFP transgene silenced by PTGS (Arabidopsis line 8z2). Using various techniques able to monitor virus infection, small and long viral RNA molecules, VSR activity, as well as phloem-mediated transport with in these lines, this study led to the identification of genes required for disease symptoms and disease symptom recovery. Moreover, the observations allowed to propose a model in which symptoms recovery occurs upon robust delivery of antiviral secondary vsiRNAs from source to sink tissues, and establishment of a vsiRNA dosage able to block the VSR activity involved in the formation of disease symptoms

Pest control strategies targeting insect olfaction represent a promising venue for control of tortricid insects (Lepidoptera: Tortricidae). Among tortricids, the grapevine moth Lobesia botrana (Denis and Schiffermüller) and the codling moth Cydia pomonella (L.) are serious pests for worldwide production of fruit crops. We employed several approaches to the olfactory system, from electrophysiological and behavioral studies in the grapevine moth, to bioinformatic and molecular studies of olfactory sensory proteins in the codling moth. At the receptor level, we studied both the Olfactory Receptors (ORs), the most common class of sensory proteins mediating detection of odors in insect antennae, and the Transient Receptor Potential (TRP) channels, a novel family of receptor, that recently were also found in the antennae of lepidopterous species. We demonstrated electrophysiological and behavioral responses of the grapevine moth to volatiles emitted by a non-host, Perilla frutescens, previously known to activate TRPs in the rat, Rattus norvegicus. In the codling moth, we characterized a novel TRP channel (TRPA pyrexia-like) and we confirmed activation of its human orthologue to the same non-host compounds active on the olfactory system of the grapevine moth. ORs were heterologously expressed in vivo and in vitro, for identification of their ligands among host and non-host plant volatiles and pheromones (deorphanization). Among several ORs of codling moth, we deorphanized a candidate pheromone receptor (PR) to plant synergists, an OR to non-host volatiles and another PR candidate to a pheromone antagonist of the insect. Our study thus opens for refinement of existing pest control, or novel applications. The behavioral response of the grapevine moth to volatiles from a nonhost plant, and the identification of a novel TRP channel in the codling moth may have perspectives for application in agriculture, targeting the somatosensory system of these tortricids. The evolutionary implications of the responses of the human orthologue of TRPA pyrexia-like to volatiles active on the grapevine moth olfactory system could imply a large degree of conservation of the receptor function. In the codling moth, identification of synergist and antagonist ligands for candidate PRs and deorphanization of an OR to non-host plant volatiles suggest a possible role of these receptors in reproductive and ecological isolation. This could lead to further refinement of existing semiochemicalbased control techniques, by enabling a better understanding of mate- and host-finding in this species.

: Les plantes ont développé au cours de leur évolution un système d’immunité innée constitué de barrières préformées et de réponses de défense induites contre les agents pathogènes. Ce travail s’inscrit dans l’étude des résistances induites chez Arabidopsis thaliana soit naturellement contre les agents pathogènes racinaires Fusarium oxysporum spp. (Fo), ou après application de phosphite (Phi), contre le parasite foliaire Hyaloperonospora arabidopsidis (Hpa). Dans la première partie du travail, les rôles des métabolites secondaires (MS) et des formes réactives de l’oxygène (ROS) dans les résistances racinaires basale et non-hôte, respectivement aux formes spéciales conglutinans (Foco) et melonis (Fom) de Fo, ont été analysés. Nous avons démontré la participation de la camalexine, une phytoalexine indolique, dans la résistance basale d’Arabidopsis à Foco. En revanche, la scopolétine, une phytoalexine phénolique, et les ROS jouent des rôles essentiels dans la résistance non-hôte à Fom. Ces données soulignent le rôle clé des MS et des ROS dans les résistances hôte et non-hôte d’Arabidopsis. Dans une deuxième partie de ce travail, le mode d’action du Phi, un oxyanion de l’acide phosphoreux (H3PO3) protégeant Arabidopsis contre l’isolat Noco2 de Hpa, a été étudié. L’effet de faibles doses de Phi est aboli chez des mutants d’Arabidopsis affectés dans la voie de transduction du signal acide salicylique (SA) indiquant que l’induction de résistance à Hpa est médiée par des mécanismes dépendants du SA. Le Phi potentialise les réponses de défense contre Hpa Noco2 via EDS1-PAD4, deux composants essentiels de la résistance basale, NPR1 et la protéine de défense PR1. L’expression de la MAP kinase MPK4, un régulateur négatif de la résistance à Hpa, est diminuée par le Phi, lors de l’inoculation par Hpa Noco2. Nos résultats démontrent que la potentialisation des réponses de défense par le Phi est associée à la régulation négative de MPK4
Plants have developed during their evolution an innate immunity system consisting of preformed barriers and induced defence responses against pathogens. This work studies resistances in Arabidopsis thaliana induced either naturally against the root pathogen Fusarium oxysporum spp. (Fo), or after application of phosphite (Phi) against the leaf pathogen Hyaloperonospora arabidopsidis (Hpa). In a first part, roles of secondary metabolites (SM) and reactive oxygen species (ROS) in basal and non-host resistances of roots to the special forms conglutinans (Foco) and melonis (Fom) of Fo, respectively, were analyzed. We demonstrated the involvement of the indolic phytoalexin camalexin, in basal resistance of Arabidopsis to Foco. In contrast, the phenolic phytoalexin, scopoletin, and ROS play essential roles in non-host resistance to Fom. These data underscore the key role of MS and ROS in basal and non-host resistances of Arabidopsis. In a second part, the mode of action of Phi, an oxyanion of phosphorous acid (H3PO3) protecting Arabidopsis against the Hpa isolate Noco2 was studied. Effect of low doses of Phi is abolished in Arabidopsis mutants affected in salicylic acid (SA) signalling, indicating that induced resistance to Hpa is mediated by SA-dependent mechanisms. Phi primes defence responses against Hpa Noco2 via EDS1-PAD4, two essential components of basal resistance, as well as NPR1 and PR1. Expression of the MAP kinase MPK4, a negative regulator of resistance to Hpa, is decreased by Phi after inoculation with Hpa Noco2. Our results demonstrate that priming of defence responses by Phi is associated with down-regulation of MPK4

碩士
國立中興大學
農業生物科技學研究所
90
Abstract Cucumber mosaic virus (CMV), infecting a very wide range of plant species (>1,000 species) is a positive-sense RNA plant virus with a tripartite genome. Based on RNA sequence homology, CMV has been divided into subgroup IA, IB and II. The CMV-2b protein which is encoded by a 3’-proximal ORF of the RNA 2 first identified in subgroup II CMV-Q and translated from a subgenomic RNA 4a. CMV-2b protein has the function in facilitating long-distance virus movement and symptom expression and has been identified as a viral counterdefence factor that interferes with the establishment of post- transcriptional gene silencing (PTGS) and salicylic acid-mediated virus resistance in plants. The 2b protein may play an important role in a wide host range of CMV. Previous, attempts by other laboratories to express the 2b protein of subgroup IA or IB in E. coli was unsuccessfully. In this work, we have successfully overexpressed the 2b protein of a subgroup IB CMV, NT9 strain, in E.coli and generated a polyclonal antibody against the 2b protein. The 2b antiserum can interact specifically with 2b protein that not only expressed in E.coli but also accumulated in NT9-CMV-infected plants. Bamboo mosaic virus (BaMV), a member of the potexvirus group with a narrow host range, infects primarily members of the Bambusoideae, few other monocotyledonous plants and Nicotiana benthamiana. In order to extend BaMV host range, we constructed BaMV derivative BaMV-2b by cloning the 2b gene of CMV NT9 strain into the BaMV vector. Using chimeric virus BaMV-2b and wild type BaMV as inocula, virus accumulation and systemic spread in the inoculated plants were compared. Our results suggest that the chimeric virus BaMV-2b engineered to express the 2b protein of CMV facilitated BaMV long distance movement in N. benthamiana (host plant) and enhanced infectivity of BaMV in N. rustica, N. tabaccum, N. samsun, Solanum tuberosum L. and Cucumis sativus L. (non-host plants).

AM plants acquire Pi via two pathways; the direct uptake pathway via plant roots and the AM pathway via external fungal hyphae and colonised cortical cells. It has been assumed that these two pathways are additive and therefore in non-responsive plants the AM pathway is often considered to be non-functional. However, data from ³²P uptake studies indicates that the AM pathway is functional in many non-responsive symbioses and in some instances supplies the majority of plant P. In recent years the high-affinity Pi transporters involved in both direct and AM Pi uptake pathways have been identified. They are expressed at the root epidermis and the symbiotic interface of colonised cortical cells and respond to the P and AM status of the plant. The overall objective of the work described in this thesis was to characterise Pi uptake via the AM pathway in barley, a non-responsive AM host, using an approach which integrated physiological measurements of plant responsiveness and AM contribution with investigations of gene expression and functional characterisation of the plant Pi transporters. A preliminary survey of field-grown barley demonstrated the persistence of AM colonisation under commercial cropping regimes in southern Australia and highlighted the relevance of AM studies to commercial agriculture. Under glasshouse conditions AM colonisation of barley induced depressions in growth and P uptake compared to NM controls. Growth depressions were unrelated to percent colonisation by two AM fungal species and could not readily be explained by fungal C demand; the strong correlation between growth and P content suggested that P was the limiting factor in these experiments. However, a compartmented pot system incorporating ³²P-labelling demonstrated that the AM pathway is functional in colonised barley and, in the interaction with G. intraradices, contributed 48% of total P. This suggested that P flux via the direct uptake pathway is decreased in AM barley. The expression of three Pi transporters, HvPT1, HvPT2 and HvPT8 was investigated in colonised roots. HvPT1 and HvPT2 have previously been localised to the root epidermis and root hairs and are involved in Pi uptake via the direct pathway whilst HvPT8 is an AM-inducible Pi transporter which was localised by in-situ hybridisation to colonised cortical cells. Using promoter::GFP gene fusions the localisation of HvPT8 to arbuscule-containing cortical cells was confirmed in living roots from transgenic barley. Quantitative real-time PCR analysis of the expression of these three Pi transporters indicated that HvPT1 and HvPT2 were expressed constantly, under all conditions regardless of AM colonisation status and indicated that decreased P flux via the direct pathway is not related to expression of these transporters. HvPT8 was induced in AM colonised roots. However, the level of expression was not related to flux via the AM pathway or arbuscular colonisation. The HvPT8 transporter was further characterised by constitutive over-expression in transgenic barley. ³²P uptake assays in excised roots demonstrated increased Pi uptake from low P solution compared to wild-type roots and confirmed that HvPT8 is a functional Pi transporter with high-affinity transport properties. This is the first report of characterisation of an AM-inducible Pi transporter in planta. When these transgenic plants were grown in solution culture there was no increase in growth or P uptake relative to wild-type or transgenic controls and growth in soil and AM colonisation were also unaffected in these transgenic lines. The data presented in this thesis highlights the importance of combined physiological and molecular approaches to characterising plant AM interactions. The persistence of AM colonisation in barley in the field indicates the importance of improving our understanding of symbiotic function in non-responsive plants. Future efforts should be directed towards understanding the signals which regulate P flux via both the direct and AM pathways with the ultimate aim of enhancing AM responsiveness of non-responsive species. Making the direct and AM pathways additive in nonresponsive species should be a key aim of future research.
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Thesis (Ph.D.) -- University of Adelaide, School of Earth and Environmental Sciences, 2008

AM plants acquire Pi via two pathways; the direct uptake pathway via plant roots and the AM pathway via external fungal hyphae and colonised cortical cells. It has been assumed that these two pathways are additive and therefore in non-responsive plants the AM pathway is often considered to be non-functional. However, data from ³²P uptake studies indicates that the AM pathway is functional in many non-responsive symbioses and in some instances supplies the majority of plant P. In recent years the high-affinity Pi transporters involved in both direct and AM Pi uptake pathways have been identified. They are expressed at the root epidermis and the symbiotic interface of colonised cortical cells and respond to the P and AM status of the plant. The overall objective of the work described in this thesis was to characterise Pi uptake via the AM pathway in barley, a non-responsive AM host, using an approach which integrated physiological measurements of plant responsiveness and AM contribution with investigations of gene expression and functional characterisation of the plant Pi transporters. A preliminary survey of field-grown barley demonstrated the persistence of AM colonisation under commercial cropping regimes in southern Australia and highlighted the relevance of AM studies to commercial agriculture. Under glasshouse conditions AM colonisation of barley induced depressions in growth and P uptake compared to NM controls. Growth depressions were unrelated to percent colonisation by two AM fungal species and could not readily be explained by fungal C demand; the strong correlation between growth and P content suggested that P was the limiting factor in these experiments. However, a compartmented pot system incorporating ³²P-labelling demonstrated that the AM pathway is functional in colonised barley and, in the interaction with G. intraradices, contributed 48% of total P. This suggested that P flux via the direct uptake pathway is decreased in AM barley. The expression of three Pi transporters, HvPT1, HvPT2 and HvPT8 was investigated in colonised roots. HvPT1 and HvPT2 have previously been localised to the root epidermis and root hairs and are involved in Pi uptake via the direct pathway whilst HvPT8 is an AM-inducible Pi transporter which was localised by in-situ hybridisation to colonised cortical cells. Using promoter::GFP gene fusions the localisation of HvPT8 to arbuscule-containing cortical cells was confirmed in living roots from transgenic barley. Quantitative real-time PCR analysis of the expression of these three Pi transporters indicated that HvPT1 and HvPT2 were expressed constantly, under all conditions regardless of AM colonisation status and indicated that decreased P flux via the direct pathway is not related to expression of these transporters. HvPT8 was induced in AM colonised roots. However, the level of expression was not related to flux via the AM pathway or arbuscular colonisation. The HvPT8 transporter was further characterised by constitutive over-expression in transgenic barley. ³²P uptake assays in excised roots demonstrated increased Pi uptake from low P solution compared to wild-type roots and confirmed that HvPT8 is a functional Pi transporter with high-affinity transport properties. This is the first report of characterisation of an AM-inducible Pi transporter in planta. When these transgenic plants were grown in solution culture there was no increase in growth or P uptake relative to wild-type or transgenic controls and growth in soil and AM colonisation were also unaffected in these transgenic lines. The data presented in this thesis highlights the importance of combined physiological and molecular approaches to characterising plant AM interactions. The persistence of AM colonisation in barley in the field indicates the importance of improving our understanding of symbiotic function in non-responsive plants. Future efforts should be directed towards understanding the signals which regulate P flux via both the direct and AM pathways with the ultimate aim of enhancing AM responsiveness of non-responsive species. Making the direct and AM pathways additive in nonresponsive species should be a key aim of future research.
Thesis (Ph.D.) -- University of Adelaide, School of Earth and Environmental Sciences, 2008

Rhinocyllus conicus Fröelich and Trichosirocalus horridus (Panzer) were introduced from Europe into North America as biological control agents of the exotic weed species Carduus nutans L. Concern exists over the feeding of these weevils on at least 25 species of native Cirsium thistles. Research was conducted to 1) estimate phenological synchrony of the eight thistle species in Tennessee with R. conicus and T. horridus, 2) investigate naturally-occurring populations of the five native Cirsium thistle species for non-target activity by R. conicus and T. horridus, 3) quantify the impacts to plants of each thistle species to feeding of R. conicus and T. horridus, and 4) identify potential areas of non-target feeding by the weevils using spatial analysis. Phenologies of two native species, C. carolinianum and C. horridulum, are synchronous with R. conicus reproduction, and all eight thistle species are phenologically synchronous with the reproduction of T. horridus. No non-target activity by R. conicus was observed in naturally-occurring populations of Cirsium thistles, but adults of T. horridus were documented for the first time on the native species C. carolinianum, C. horridulum, and C. muticum. In caged plant studies, larvae of R. conicus completed development in heads of C. carolinianum and C. horridulum and reduced seed numbers of both native species. Basal meristems of all eight thistle species exposed to T. horridus were damaged at varying levels in caged plant studies, but no other impacts to plants were observed. Spatial analyses showed associations between Mahalanobis distance values and plant counts of Carduus nutans and Cirsium carolinianum in predicted habitats, and the occurrence of Carduus nutans was associated with the occurrence of both weevil species in these habitats. About 12% of the total study area consisted of habitats where C. nutans and Cirsium carolinianum overlap. The potential exists for these weevils to utilize native Cirsium species found in Tennessee as plant hosts. The spatial model developed during this study not only allows potential monitoring of populations of C. carolinianum to be more targeted, but also may be modified to apply to other systems involving interactions among introduced and native species.

The production of cereal crops is essential to secure a future that feeds the continuously growing population. Rust fungi reduce host fitness by feeding on their living tissue and interfere with the global production of crops. Cereal rusts, like Puccinia hordei (the causal agent of barley leaf rust) and Puccinia triticina (the causal agent of wheat leaf rust), have a narrow host range and colonize only one particular species. The most durable type of resistance, non-host resistance (NHR), is the immunity of an entire plant species to all strains of a pathogen species. Exploring the genetics of NHR has proven to be challenging because most interspecific hybrids are infertile. Previously, barley Rphq2 and Rph22, which encode orthologous lectin receptor-like kinases (LecRKs), were transformed into an experimental barley line, Golden SusPtrit, and showed resistance against adapted and non-adapted leaf rust species. We used these transgenic barley lines in the current project to explore the effect of the LecRKs on four wheat leaf rust (P. triticina) isolates. We used the settling tower method to inoculate four isolates of P. triticina on Rphq2 and Rph22 transgenic families. We found that most transgenic families showed an increase in resistance compared to the non-transgenic control 750-E1. By measuring the infection frequency of the infections, we identified that P. triticina isolates 93012 and 95012 had opposite virulence effects on two barley families, Rphq2-E5 and Rph22-E2A. Although the expression levels of Rphq2 and Rph22 followed an induction trend, we did not find significant differences between the isolates. We conclude that resistance mediated by Rphq2 and Rph22 against P. triticina isolates does not involve an isolate-specific component. Thus, we propose investigating differences between rust species to further explore the molecular aspect of non-host resistance.

Fusarium oxysporum (Fo) is a soil-borne fungal pathogen that causes vascular wilt disease on a broad range of plants, including agricultural crops and the model plant Arabidopsis thaliana. There are non-pathogenic members of the Fo species complex that confer defense benefits against other pathogens to the host plant, however alteration to the host’s physiology through interaction with one of these strains, Fo47, have not been described. In this study, we aimed to establish the Fo47-A. thaliana interaction and determine if Fo47 reduces disease severity of a pathogenic Fo isolate, Fo5176. Additionally, we sought to use bioinformatics to mine transcriptomic data of the infection between Fo47 and A. thaliana for putative effectors from the non-pathogenic isolate using a pipeline that is validated by identifying known effectors in the interaction between Fo5176 and A. thaliana. Phenotypic characterization of A. thaliana plants inoculated with Fo47 or Fo5176 has revealed a significant increase in rosette biomass of Fo47 inoculated plants when compared to mock (sterile water) inoculated plants. As is observed in other systems, treatment of plants with Fo47 prior to challenging with pathogenic Fo significantly reduces the disease severity over time. The results of this study suggest that Fo47 is a possible biocontrol agent against Fo5176, and that inoculation with non-pathogenic Fo alters the physiology of A. thaliana such that it has a higher rosette biomass without alterations to the water status of the plant. Our pipeline for extracting putative effectors using transcriptomic data as a critical filter generated 13 candidate genes for further experimentation to determine their role in the Fo47-A. thaliana interaction. This research reports the first known observation that Fo47 increases the shoot biomass of the host plant it is interacting with, and that the model plant A. thaliana can be used as a host to examine the spectrum of interactions capable within the Fusarium oxysporum species complex.