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    Studies on bacterial speck of tomatoes caused by Pseudomonas syringae pv tomato : a thesis presented in fulfilment of a Masterate of Science by thesis only at Massey University
    (Massey University, 1980) Pyke, Nicholas Brian
    The taxonomy of the causal agent of bacterial speck of tomatoes is discussed and the trinomial Pseudomonas syringae pathovar tomato (Okabe) Young, Dye and Wilkie is adopted. A vacuum infiltration method of artificially inoculating seed was used and P.syringae pv tomato was detected in both artificially and naturally infested seed using sensitive enrichment culture techniques. The pathogen can remain viable between seed harvest and sowing in association with seed but seed-plant transfer was only occasionally demonstrated. The acid seed extraction method and other germicidal seed treatments were evaluated for their effect on the seedborne pathogen. Streptomycin sulphate as a slurry treatment (2.5g a.i./Kg of seed) just prior to seed sowing was the only totally effective seed treatment tested. The potential for survival in infected crop debris, soil and on alternative hosts was shown. However, the pathogen was not isolated from weeds in infected tomato crops and no conclusive evidence of systemic infection was found.
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    Investigation of the biosynthesis of exopolysaccharides within the biofilm matrix of Pseudomonas aeruginosa and Pseudomonas syringae pv. actinidiea : a thesis presented in partial fulfilment of the requirements for degree of Doctor of Philosophy in Microbiology and Genetics at Massey University, Manawatu, New Zealand
    (Massey University, 2017) Ghods, Shirin
    Polysaccharides are highly abundant natural biopolymers, which have biologically significant structural functions in living organisms. Various polysaccharides, with specific physicochemical properties, contribute to biofilm formation; defined as cell aggregations surrounded by extracellular polymeric substances. They are also important in the context of bacterial pathogenesis, while some have been harnessed for industrial and biomedical applications due to their unique chemical compositions and properties. In present study, we aimed at studying biofilm formation by Pseudomonas aeruginosa and P. syringae pv. actinidiae, respectively known as human and plant pathogens. In this context we focused on the production of exopolysaccharides, which predominantly constitute the biofilm matrix of these pathogenic bacteria. Here, we uncovered that the polysaccharide isolated from P. syringae pv. actinidiae biofilm mainly consists of rhamnose, fucose and glucose and it was cautiously introduced as a novel polysaccharide. In the context of disease control, and developing a management program, we provided some evidences for the effectiveness of chlorine dioxide and kasugamycin in the control of the bacteria living in both biofilm and planktonic modes. Furthermore, we investigated alginate biosynthesis as major polysaccharide contributing to mucoid biofilm formation by P. aeruginosa. We generated various mutants producing a variety of alginates with different chemical compositions. Also, this enabled us to analyse functional relationships of protein subunits involved in multiple steps of alginate biosynthesis including alginate polymerization, modification and secretion. We present evidence that while alginate unravelled that while alginate is polymerised and translocated across the membrane by a multiprotein complex, acetylation and epimerisation events positively and negatively correlated with the polymerization of the alginate or molecular mass, respectively. Analysis of the biofilms showed that biofilm architecture and cell-to-cell interactions were differently impacted by various compositions of the alginates. Also, this study provided insights into the c-di-GMP mediated activation of alginate polymerization upon binding to c-di-GMP as well as assigning functional roles to Alg8 and Alg44 including their subcellular localization and distribution. Here, we also used current knowledge of the alginate biosynthesis pathway to assess the production of alginate from biotechnologically accepted heterologous hosts including Escherichia coli and Bacillus megaterium strains. Primarily, we evaluated the production and functionality of the minimal protein requirements in nonpathogenic heterologous hosts, required for producing alginate precursor, and proceeding into polymerization and secretion steps. Overall, we concluded that polysaccharides play a major role in the formation of bacterial biofilms while chemical composition is a key determinant for biofilm architecture and development. This contribution to understanding the biosynthesis of bacterial polysaccharides and their properties could provide the necessary knowledge not only for developing novel therapeutics, but also for harnessing such biopolymers for various industrial applications and production via biotechnological procedures.
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    Identification and functional analysis of Pseudomonas syringae pv. actinidiae effector-triggered immunity in Nicotiana spp. and Arabidopsis thaliana : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Plant Science at Massey University, Manawatu, New Zealand
    (Massey University, 2017) Choi, Sera
    Pseudomonas syringae pv. actinidiae (Psa) is the causal agent of bacterial canker in commercially important cultivars of kiwifruit (Actinidia delicosa and A. chinensis) worldwide, including New Zealand. Like many gram-negative pathogens, Psa is expected to utilise type III effectors to promote virulence in host plants. In order to better understand Psa effector-triggered immunity and susceptibility, we aimed to investigate multiple molecular characteristics of Psa type III effectors and their recognition mechanisms in model plants, Nicotiana spp. and Arabidopsis thaliana. Nicotiana tabacum and N. benthamiana are widely-used model plants for Agrobacterium-mediated transient expression (agroinfiltration) of effectors for functional characterization. Firstly, we screened multiple characteristics of effectors from two Psa strains, Psa NZ V13 and Psa NZ LV5. The former is a strongly virulent and the latter is a weakly virulent strain in kiwifruit. By using agroinfiltration in Nicotiana spp. to express individual effector proteins, we observed diverse subcellular localisation for Psa effectors. Additionally, we identified multiple Psa effectors that can trigger HR-like cell death (HCD) in both N. tabacum and N. benthamiana. Using virus-induced gene silencing (VIGS), we identified that some Psa effector-triggered HCD requires the immunity regulator SGT1, suggesting that the Psa effector-triggered HCD could be a result of immunity activation. We focused on one Psa NZ V13 effector, HopZ5, which belongs to the YopJ-like acetyltransferase family. HopZ5 triggers hypersensitive response (HR) in Arabidopsis accession, Ct-1. Another Arabidopsis accession, Col-0, does not develop an HR but shows immunity in response to HopZ5. The gene that confers HopZ5-triggered HR in Ct-1 was identified as SOBER1 (SUPPRESSOR OF AVRBST-ELICITED RESISTANCE 1) by using recombinant inbred lines derived from two parental accessions, Ct-1 and Col-0. SOBER1 is a known suppressor of Xanthomonas effector AvrBsT-triggered immunity. Interestingly, AvrBsT also belongs to YopJ family. Uniquely, SOBER1 specifically suppressed HCD triggered by several YopJ-like acetyltransferase effectors in N. benthamiana, including HopZ5 and HopZ3 from Psa. This suggests a common mechanism shared between a subset of YopJ-like acetyltransferase effectors is suppressed by SOBER1. Finally, we identified one Arabidopsis accession, Ga-0, which carries a truncated SOBER1 variant but does not develop an HR upon HopZ5 delivery. Using bulked- segregant analysis of an F2 population derived from a cross between Ct-1 and Ga-0, we mapped the locus conferring HopZ5-recognition in Ct-1 to the upper arm of Chromosome 3.
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    Effector-triggered immunity against Pseudomonas syringae pv. actinidiae in nonhost plants : thesis submitted to the Massey University for the degree of Doctor of Philosophy
    (Massey University, 2017) Jayaraman, Jay
    Pseudomonas syringae pv. actinidiae (Psa) is a virulent and highly damaging pathogen causing bacterial canker in all currently commercially important cultivars of kiwifruit (Actinidia spp.). Arabidopsis and Nicotiana spp. plants, however, are nonhosts to Psa. In our course of investigating the various nonhost resistance mechanisms in play against Psa, we identified several sources of resistance against several Psa strains as well as a possible novel virulence mechanism used by Psa and Hyaloperonospora arabidopsidis (Hpa), a b iotrophic pathogen of Arabidopsis. Firstly, we discovered that the highly virulent strain, Psa V13, triggers hypersensitive response (HR) in Arabidopsis in an accession-­‐specific manner and that HopZ5PsaV13, a member of the YopJ family of putative acetyltransferases, confers this bacterial avirulence. We also show that the immunity triggered by HopZ5 is independent from HR in the Arabidopsis accession Col-­‐0. Through mutagenesis, we show that key amino acid residues predicted for acetyltransferase activity are vital to HopZ5-­‐triggered immunity and HR, phenotypes reproduced in Nicotiana spp. Secondly, we identified multiple sources of avirulence for the kiwifruit low-­‐ virulence strain, Psa LV5, in Arabidopsis and Nicotiana benthamiana, namely homologs of previously characterized effectors, HopAR1 and HopAB3, respectively. We additionally show that HopAB3 can trigger resistance in cultivated tomato putatively due to a novel recognition by a cultivated tomato homolog (SlPtoB) of the resistance gene Fen. Finally, we identified several nuclear-­‐localized effectors from Psa and Hpa that interact with Arabidopsis WRKY transcription factors, different to WRKYs targeted by previously identified AvrRps4 and PopP2. We show that some WRKYs can trigger a cell death response in N. benthamiana when overexpressed and that coexpression of AvrRps4 or PopP2 is able to suppress this cell death response for the WRKYs they interact with. We show that this suppression is associated with suppression of transcriptional activation ability of the WRKY and 7 propose that this mechanism of transcription suppression may be utilized by other Psa and Hpa effectors identified in this study.
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    The role of integrative conjugative elements in evolution of the kiwifruit pathogen Pseudomonas syringae pv. actinidiae : a thesis submitted in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Genetics at Massey University, Albany Campus, New Zealand
    (Massey University, 2017) Colombi, Elena
    Horizontal gene transfer (HGT) is a major force driving evolution in rokaryotes. Among the different contributions to bacterial fitness, HGT underpins the evolution of pathogenicity and virulence in several bacterial pathogens. Pseudomonas syringae pv. actinidiae (Psa) first emerged as a pathogen of Kiwifruit in Asia during the 1980’s. In 2008 an outbreak occurred in Italy that rapidly spread to major kiwifruit growing areas of the world. During its global journey the outbreak lineage independently acquired divergent Integrative and Conjugative Elements (ICEs), harbouring an identical set of cargo genes that are hypothesized to be involved in the plant-­‐pathogen interaction. Here I show that the three ICEs acquired by Psa belong to a diverse family of ICEs present only in plant-­‐associated Pseudomonas ssp. (the PsICEs). The evolution of PsICEs is characterized by extensive inter-­‐ICE recombination events that are frequent enough to mask evolutionary history, producing chimeras with variable patterns of similarity to each other, yet maintaining a syntenic backbone where cargo genes are integrated in conserved positions. Although there are different lasses of PsICE cargo genes, one set was frequently recovered: those contained on a Tn6212 element. Tn6212 confers a selective benefit when Psa is grown on succinate, fumarate, or malate as the only carbon source, but no phenotype was detected in planta. Members of the PsICE family also confer copper resistance to Psa strains isolated in New Zealand. I analyzed a number of these and showed transfer in vitro and in planta. I also measured the fitness consequences of ICE carriage, captured the de novo formation of novel recombinant ICEs, and explored ICE host-­‐range. Together my work, which began with observations from genome sequences before moving to experimental studies in the laboratory, has provided new insights into the role that horizontal gene transfer plays in the evolution of virulence.
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    Kiwifruit bacterial canker in 'Hayward' kiwifruit : the application of observational study design and epidemiological techniques to the study of disease outbreaks affecting plant health : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Veterinary Epidemiology, Institute of Veterinary, Animal and Biomedical Sciences at Massey University, Manawatu, New Zealand
    (Massey University, 2017) Froud, Karyn Janine
    Bacterial canker of kiwifruit, caused by Pseudomonas syringae pv. actinidiae (Psa) biovar 3, was first recorded in New Zealand in November 2010 and quickly made production of the goldfleshed kiwifruit cultivar, ‘Hort16A’, which is highly susceptible to Psa, no longer viable in the Bay of Plenty region. Production of the green-fleshed cultivar, ‘Hayward’ has remained viable but there is uncertainty around its long-term productivity. This thesis investigated aspects of Psa in commercial ‘Hayward’ orchards using observational studies. The aims were to: 1) quantify a change in productivity associated with disease; 2) determine the prevalence of disease in orchards; 3) identify factors that altered the initial development of disease and 4) identify factors that impact on the presence of severe disease. Severe disease was defined as 5% or more female vines in a block showing the systemic symptoms of green shoot wilt and cane dieback. To determine Psa effects on productivity historical data from 2599 ‘Hayward’ orchards were analysed. No reduction in productivity was found until 1 year after initial detection of Psa, after controlling for other orchard inputs that affect productivity. A crosssectional survey was sent to all Psa confirmed ‘Hayward’ orchards and 430 growers provided information about one of their ‘Hayward’ orchard blocks. The survey found 84% of orchard blocks were affected by disease and 57% had green shoot-wilt and/or cane dieback reported. Blocks typically had a low within block prevalence of systemic symptoms (Median = 5% of vines). In 194 orchards that were asymptomatic at the start of the study period the probability of disease developing in a block increased in association with use of Psa protectant sprays immediately post-pruning and using artificial pollination. A lower probability of disease developing was associated with undertaking summer girdling and with the presence of older male vines. The probability of developing severe disease was investigated in 331 orchard blocks that were symptomatic. The probability increased with time after Psa was first detected in a block and was highest when frost damage occurred, when poplar, cypress or pine shelter belts were present and when artificial pollination was used. The probability of severe bacterial canker was lower when spring girdling of female vines was undertaken. The results of this study can be used to prioritise future research. The thesis has also demonstrated the utility of observational studies for plant disease research.