Massey Documents by Type

Permanent URI for this communityhttps://mro.massey.ac.nz/handle/10179/294

Browse

Subject: search.filters.subject.Dothistroma needle blight

Search Results

Now showing 1 - 5 of 5
  • Thumbnail Image
    Item
    Development of a rapid loop-mediated isothermal amplification assay for the detection of Dothistroma septosporum
    (MDPI (Basel, Switzerland), 2021-03-19) Myrholm CL; Tomm BD; Heinzelmann R; Feau N; Hamelin RC; McDougal R; Winkworth RC; Ramsfield TD; Moricca S; Panzavolta T
    A Loop-Mediated Isothermal Amplification (LAMP) assay was developed for the detection of the pine pathogen Dothistroma septosporum (G. Dorog.) M. Morelet. The specificity of the LAMP assay was tested using a selection of pine needle fungi, including Dothistroma pini Hulbary, and Lecanosticta acicola (Thüm.) Syd.; only D. septosporum DNA was amplified by the test. In terms of sensitivity, the assay was able to detect as little as 1 pg of total D. septosporum DNA. This assay enables DNA extracted from diseased host needles to be rapidly tested for the presence of D. septosporum using relatively simple to operate equipment away from a fully equipped molecular biology laboratory.
  • Thumbnail Image
    Item
    Secondary metabolism of the forest pathogen Dothistroma septosporum : a thesis presented in the partial fulfilment of the requirements for the degree of Doctor of Philosophy (PhD) in Genetics at Massey University, Manawatu, New Zealand
    (Massey University, 2016) Ozturk, Ibrahim Kutay
    Dothistroma septosporum is a fungus causing the disease Dothistroma needle blight (DNB) on more than 80 pine species in 76 countries, and causes serious economic losses. A secondary metabolite (SM) dothistromin, produced by D. septosporum, is a virulence factor required for full disease expression but is not needed for the initial formation of disease lesions. Unlike the majority of fungal SMs whose biosynthetic enzyme genes are arranged in a gene cluster, dothistromin genes are dispersed in a fragmented arrangement. Therefore, it was of interest whether D. septosporum has other SMs that are required in the disease process, as well as having SM genes that are clustered as in other fungi. Genome sequencing of D. septosporum revealed that D. septosporum has 11 SM core genes, which is fewer than in closely related species. In this project, gene cluster analyses around the SM core genes were done to assess if there are intact or other fragmented gene clusters. In addition, one of the core SM genes, DsNps3, that was highly expressed at an early stage of plant infection, was knocked out and the phenotype of this mutant was analysed. Then, evolutionary selection pressures on the SM core genes were analysed using the SM core gene sequences across 19 D. septosporum strains from around the world. Finally, phylogenetic analyses on some of the SM core genes were done to find out if these genes have functionally characterised orthologs. Analysis of the ten D. septosporum SM core genes studied in this project showed that two of them were pseudogenes, and five others had very low expression levels in planta. Three of the SM core genes showed high expression levels in planta. These three genes, DsPks1, DsPks2 and DsNps3, were key genes of interest in this project. But despite the different expression levels, evolutionary selection pressure analyses showed that all of the SM core genes apart from the pseudogenes are under negative selection, suggesting that D. septosporum might actively use most of its SMs under certain conditions. In silico predictions based on the amino acid sequences of the proteins encoded by SM core genes and gene cluster analyses showed that four of the SM core genes are predicted to produce known metabolites. These are melanin (DsPks1), cyclosporin (DsNps1), ferricrocin (DsNps2) and cyclopiazonic acid (DsHps1). Gene cluster analyses revealed that at least three of the D. septosporum SMs might be produced by fragmented gene clusters (DsPks1, DsNps1, DsNps2). This suggested that dothistromin might not be the only fragmented SM gene cluster in D. septosporum. According to phylogenetic analyses, some of the D. septosporum SM core genes have no orthologs among its class (Dothideomycetes), suggesting some of the D. septosporum SMs may be unique. One such example is the metabolite produced by DsNps3. Comparison of wild type and ΔDsNps3 D. septosporum strains showed that the ΔDsNps3 strain produces fewer spores, less hyphal surface network at an early stage of plant infection, and lower levels of fungal biomass in disease lesions compared to wild type, suggesting that the DsNps3 SM may be a virulence factor. Attempts to identify a metabolite associated with DsNps3, and to knockout another gene of key interest, DsPks2, for functional characterization were unsuccessful. Further work is required to confirm the gene clusters, characterise the SMs and their roles. However, the findings so far suggest that dothistromin is unlikely to be the only D. septosporum SM that is a virulence factor in since the DsNps3 SM also appears to be involved in virulence. Likewise the fragmented dothistromin cluster may not be the only one in the genome and there may be at least three more fragmented SM gene clusters.
  • Thumbnail Image
    Item
    Investigation of dothistroma needle blight development on Pinus radiata : a thesis presented in the partial fulfilment of the requirements for the degree of Doctor of Philosophy (PhD) in Microbiology and Genetics at Massey University, Manawatu, New Zealand
    (Massey University, 2014) Kabir, Md Shahjahan
    Dothistroma needle blight (DNB), caused by the fungi Dothistroma septosporum and Dothistroma pini, is an important foliar disease of pine species throughout the world and predictions of the future spread of this disease have been made using climate models. Although DNB infection is prevalent in many forests, attempts to achieve infection under controlled laboratory or glasshouse conditions are notoriously difficult. However, artificial infection is a very important tool for studying different aspects of plant-microbe interactions, such as pathogen life style and roles of virulence factors. D. septosporum was thought to have a hemi-biotrophic life style but this was not formally investigated in planta. The non-host selective toxin dothistromin produced by this fungus was shown not to be essential for pathogenicity but its role in pathogen virulence was unknown. The aims of this study were to improve the DNB pathogenicity assay and to use this system to test the hypotheses that D. septosporum is a hemi-biotrophic pathogen and that dothistromin plays a role in virulence. A new sporulation medium (pine needle medium with glucose) was used to obtain sufficient viable D. septosporum spores. The critical microclimatic component of leaf wetness was optimised to have a short (4-7 d) high wetness period followed by 'medium' wetness (continual misting), and using these conditions >80% needle infection was routinely achieved on Pinus radiata seedlings. A combination of microscopy, biochemical and molecular studies over a timecourse of infection of P. radiata by D. septosporum confirmed its hemi-biotrophic life style. Restricted mesophyll colonisation, shorter lesions and fewer spores from P. radiata needles infected with dothistromin-deficient mutants, compared to those with wild type D. septosporum, suggested that dothistromin has a role in virulence. Interestingly ‘green islands’ in which chlorophyll levels were maintained at higher levels than adjacent chlorotic and necrotic regions, surrounded early-appearing lesions caused by both wild-type and mutant isolates. At a later developmental stage of the lesion the green islands were still present in the mutant but appeared to be masked by the extended dothistromin-containing lesions in the wild type, which lead to the hypothesis that chloroplasts could be a site of action of dothistromin. The discovery that dothistromin is a virulence factor opens up new insights into the Dothistroma-pine interaction. This fundamental finding will be useful for management strategies for this important disease in the future.
  • Thumbnail Image
    Item
    Investigations of dothistromin gene expression in Dothistroma septosporum and the putative role of dothistromin toxin : a thesis presented in the partial fulfilment of the requirements for the degree of Doctor of Philosophy (PhD) in Molecular Biology at Massey University, Palmerston North, New Zealand.
    (Massey University, 2007) Schwelm, Arne
    Dothistroma septosporum causes pine needle blight, a foliar disease currently causing epidemics in the Northern hemisphere. D. septosporum synthesizes dothistromin, a mycotoxin similar in structure to the aflatoxin (AF) precursor versicolorin B. Orthologs of AF genes, required for the biosynthesis of dothistromin, have been identified along with others that are speculated to be involved in the same pathway. The dothistromin genes are located on a mini-chromosome in Dothistroma septosporum but, in contrast to AF genes, not in a continuous cluster. The aim of this study was to increase knowledge of the biological role of dothistromin, which was previously a suspected pathogenicity factor. To identify putative roles of dothistromin, the dothistromin gene expression was investigated and green fluorescence protein (GFP) reporter gene strains of D. septosporum were developed. Expression analyses of dothistromin genes revealed co-regulation. More surprisingly, dothistromin is produced at an early stage of growth and gene expression is highest during exponential growth. This is fundamentally different to the late exponential/stationary phase expression usually seen with secondary metabolites such as AF. Strains with a dothistromin gene (dotA) promoter-regulated GFP confirmed early expression of the toxin genes, even in spores and germtubes. Parallel studies with transformants containing a GFP-DotA fusion protein suggest spatial organization of dothistromin biosynthesis in intracellular vesicles. The early expression of dothistromin genes led to the hypotheses that dothistromin is either required in the early stage of the plant/fungi interaction, or for inhibiting the growth of competing fungi. Constitutive GFP strains helped to determine that dothistromin is not a pathogenicity factor. However, a putative role of dothistromin in competition with other fungi, including pine-colonizing species, was detected, supporting the second hypothesis. It was shown that dothistromin-producing strains appear to have a competitive advantage which is lacking in dothistromin-deficient strains. However, some competitors were not affected and have potential as biocontrol agents. In summary, this work has led to the discovery of an unusual pattern of regulation of a secondary metabolite, has made substantial progress in identifying the biological role of dothistromin, and has indicated potential for biocontrol of Dothistroma needle blight.
  • Thumbnail Image
    Item
    Targeted Gene Mutations in the Forest Pathogen Dothistroma septosporum Using CRISPR/Cas9.
    (8/04/2022) McCarthy HM; Tarallo M; Mesarich CH; McDougal RL; Bradshaw RE
    Dothistroma needle blight, caused by Dothistroma septosporum, has increased in incidence and severity over the last few decades and is now one of the most important global diseases of pines. Disease resistance breeding could be accelerated by knowledge of pathogen virulence factors and their host targets. However, this is hindered due to inefficient targeted gene disruption in D. septosporum, which is required for virulence gene characterisation. Here we report the first successful application of CRISPR/Cas9 gene editing to a Dothideomycete forest pathogen, D. septosporum. Disruption of the dothistromin pathway regulator gene AflR, with a known phenotype, was performed using nonhomologous end-joining repair with an efficiency of > 90%. Transformants with a range of disruption mutations in AflR were produced. Disruption of Ds74283, a D. septosporum gene encoding a secreted cell death elicitor, was also achieved using CRISPR/Cas9, by using a specific donor DNA repair template to aid selection where the phenotype was unknown. In this case, 100% of screened transformants were identified as disruptants. In establishing CRISPR/Cas9 as a tool for gene editing in D. septosporum, our research could fast track the functional characterisation of candidate virulence factors in D. septosporum and helps set the foundation for development of this technology in other forest pathogens.