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    Identification and characterization of effector proteins from pine needle pathogens : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy (PhD) in Genetics at Massey University, Manawatū, New Zealand
    (Massey University, 2022) Massoco Tarallo, Mariana
    Collectively, Dothistroma septosporum, Cyclaneusma minus and Phytophthora pluvialis cause serious foliar diseases on Pinus radiata in New Zealand and on many other pine species worldwide. Considering the ecological and economic importance of forest trees, understanding how these pathogens interact with their hosts on a molecular level is critical as it could lead to new and durable approaches to control the diseases they cause. Pathogens have the ability to deliver proteinaceous virulence factors, termed effectors, into the apoplast and cell cytoplasm of their host plants. Effectors typically promote host colonization through suppression of the plant immune system. However, in resistant host plants, one or more of these effectors can be recognized by corresponding immune receptors to activate the plant immune system. Often, one of the main outputs of this immune system is a localised cell death reaction, termed the hypersensitive response (HR), which renders the pathogen unable to cause disease (avirulent). The general goal of this thesis was to identify shared candidate effector (CE) proteins between the three foliar pine pathogens and to characterise their virulence (or avirulence) functions. This is important because disease resistance based on core effectors that are vital for a pathogen’s ability to cause disease is more likely to be durable. Using a combination of “omics” information and bioinformatic tools, two sets of orthologous CE proteins were identified between D. septosporum, C. minus and P. pluvialis, while several other sets were identified between the two fungal pathogens. Some of these CEs had the ability to trigger cell death responses in non-host Nicotiana plants, and some were shown to activate Nicotiana benthamiana genes involved in pathogen-associated molecular pattern-triggered immunity and HR. CEs were also screened in the host, Pn. radiata, using a method developed in this thesis, where it was determined that some of these CEs also trigger cell death. Two conserved cell death elicitor families, Ecp20 and Ecp32, were identified from D. septosporum and its close relative Fulvia fulvum, and the cell death triggered by some family members in N. benthamiana was shown to require membrane-localized receptor-like proteins. Tertiary structure predictions of CEs provided insights into the possible roles and host targets of these proteins during pine infection. Moreover, a shared β-trefoil fold was found between sequence-unrelated CE proteins from the three pine pathogens, along with evidence that they are also present in many other fungal species. A CRISPR/Cas9 gene editing methodology was applied to D. septosporum for the first time, which allowed for the functional characterization of three D. septosporum CE genes, two of which are also present in C. minus and P. pluvialis. Collectively, this thesis provides a significant advance in our understanding of pine-pathogen interactions at the molecular level and provides a blueprint for similar studies in other forest pathosystems.
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    Effector delivery and effector characterisation in Dothistroma needle blight of pines : a dissertation presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy (Sciences) in Genetics/Molecular Plant Pathology at Massey University, Manawatū, New Zealand
    (Massey University, 2018) Hunziker, Lukas
    The filamentous fungus Dothistroma septosporum causes a serious foliar disease, Dothistroma needle blight (DNB), on Pinus radiata in New Zealand and on many pine species worldwide. Potentially correlated to changes in climate, this disease has been on the rise for 20 to 30 years, and current countermeasures often struggle to contain the damage it causes. A molecular approach to combat DNB could be promising. Effectors are small proteins secreted by pathogens to promote host colonisation, and have been a major focus of plant pathologists in recent years. However, effector biology in pathogens of gymnosperms has received little research attention. Here, candidate effectors (CEs) were selected using a series of computational prediction tools, as well as RNAseq data from a compatible D. septosporum-pine interaction. A shortlist of 55 highly in planta expressed CEs, predicted to be secreted to the apoplast, was characterised in silico. While almost half of them lacked a predicted function, none were exclusive to D. septosporum. Seventeen effector candidates of particular interest were taken forward for functional characterisation. Specifically, these proteins were screened for induction of plant defences in the form of cell death in the model plants Nicotiana benthamiana and N. tabacum using an Agrobacterium transient expression assay. Five CEs induced cell death in these plants, suggesting recognition by the plant defence machinery. Of those five, three are similar to previously described proteins. Effector screening methods are not available for pine, thus various approaches to achieve this were trialled. A high-throughput method to collect each protein in the apoplastic wash fluid of N. benthamiana was developed. This fluid was applied to P. radiata shoots raised from tissue culture to screen for a response, with promising results. Along with an array of D. septosporum CEs, these shoots may ultimately be used to screen for resistant pine genotypes. Selection of genotypes at this early stage could speed up DNB resistance screening for pine breeding and the protocol could be transferred to related pathosystems. This research also contributes to the molecular understanding of forest diseases and effectors that may be common among pathogens of distantly related hosts.
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    Genetic diversity and gene expression analysis of Phytophthora pluvialis, a foliar pathogen of conifers : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy (PhD) in Genetics, Massey University, Manawatū, New Zealand
    (Massey University, 2018) Brar, Simren
    Phytophthora pluvialis is the causal agent of red needle cast on Pinus radiata in New Zealand. It was first isolated in 2008 but had previously been recovered from tanoak (Notholithocarpus densiflorus) and Douglas fir (Pseudotsuga menziesii) trees in Oregon, USA in 2002. Phytophthora pluvialis was subsequently described as a new species in 2013 and classified as a clade 3 Phytophthora species. The aims of this study were to (1) gain a better understanding of the genetic diversity and population structure of P. pluvialis and (2) examine gene expression profiles of P. pluvialis from naturally infected P. radiata seedlings. Studying the genetic diversity and population structure of P. pluvialis provided insight into the mode of reproduction of this pathogen and helped determine if P. pluvialis was introduced into New Zealand. This information is also important for the development of management strategies for P. pluvialis. Twenty-seven single nucleotide polymorphism (SNP) markers were designed to genotype a total of 360 isolates of P. pluvialis collected from New Zealand and the USA. The genotypic data showed that the population in New Zealand has lower diversity than the USA population. A minimum spanning network (MSN) showed two unique clusters in the New Zealand population, suggesting there may have been two separate introductions of P. pluvialis. For the second study, samples were collected from 45 P. radiata grafted plants that were part of a field trial, with the aim of identifying genes that are highly expressed and may be important for virulence. Interestingly, Phytophthora kernoviae was found in more of the samples than P. pluvialis. Needle samples were collected, RNA was extracted and sequenced, and the normalised reads that mapped to the genome of P. pluvialis were compared to those from P. pluvialis grown in culture. Differentially expressed genes (DEGs) of P. pluvialis that showed higher expression in the field trial included potential orthologs of sugar transporter, GH12 and effector genes with known pathogenicity functions in other species. This is the first study to examine the genetic diversity of P. pluvialis in New Zealand and the USA., and to examine the gene expression of a Phytophthora forest pathogen in the field. The results from these studies provide useful tools for forest disease management. The SNP markers can be used to monitor the population of P. pluvialis in New Zealand. The highly expressed genes can be used to help identify resistance genes in P. radiata that can be incorporated into future breeding programs.
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    The predictive ability of seven sigmoid curves used in modelling forestry growth : a thesis submitted to the Institute of Information Sciences and Technology in partial fulfilment of the requirements for the degree of Master of Applied Statistics at Massey University, February, 2000
    (Massey University, 2000) Lee, Anthony
    In this thesis we study seven sigmoid growth curve families to determine which best fit pinus radiata basal area against age data. We fit the sigmoid models to the data of distinct plots rather than the pooled data of sets of plots. The seven growth models are the three-parameter Chapman-Richards, Hossfeld, Schumacher, Weibull and Gompertz models and the four-parameter Levakovic and Sloboda models. This investigation was inspired by Dr. Richard Woollons' observation that sigmoid curves vary consistently in their estimation of the asymptote, and those functions giving bigger asymptotes have better goodness-of-fit properties. It is shown that models with better goodness-of-fit properties are better predictors of basal area. This indicates that the three-parameter Chapman-Richards model and the four-parameter Levakovic and Sloboda models are superior to the Hossfeld, Schumacher, Weibull and Gompertz models. We do however recommend caution in the use of the Levakovic model where convergence of the nonlinear least squares algorithm is often difficult. Also, parameter-effects curvature of the four-parameter models was on occasion seen to be unacceptably large and we recommend careful examination of curvature in the selection of a candidate function. We demonstrate that the Schumacher model predicts larger asymptotes than the other models but do not conclude that this model has better goodness-of-fit properties, contrary to Dr. Woollons' observation. We do however conclude that models with better goodness-of-fit do have better predictive power. The study of the growth curves is divided into six parts. Firstly, we investigate fundamental properties of the growth curves with particular attention paid to the point of inflection and the asymptote. Secondly, the models are fitted to pinus radiata data and goodness-of-fit properties are investigated. Additionally, we discuss practical considerations required when fitting these models using nonlinear least squares; in particular the location of starting values and reparameterization of the growth models. We note the ease of fitting the Chapman-Richards model and the relative difficulty in fitting the Levakovic and Sloboda models. Thirdly, we empirically demonstrate that the Schumacher model has the largest asymptote. Fourthly, we investigate the robustness of the models in predicting basal area using both pinus radiata data and simulated data. Next, Padé rational approximations of the growth curves are investigated to begin a theoretical study of the observed behaviour of the fitted growth curves in an attempt to explain the goodness-of-fit and predictive power of the curves by providing a common basis of comparison. A possible additional area of research is indicated by this analysis - the estimation of properties at the point of inflection and the fitting of the associated rational function to the data. Finally, the results in preceding chapters are summarised to rank the growth curves according to their effectiveness in modelling pinus radiata growth data. We do not conclude that one model is optimal in all respects but do give a hierarchy of suitability, with the Chapman-Richards model at the top of this hierarchy.
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    Vigour assessment in Pinus radiata D. Don seeds : a thesis presented in partial fulfilment of the requirements for the degree of Master of Agricultural Science in seed technology at Massey University, Palmerston North, New Zealand
    (Massey University, 1990) Kartiko, Hero Dien P. (Hero Dien Pancang)
    The sensitivity and/or predictivity of various vigour test methods (which include conductivity, tetrazolium, x-ray contrast, seedling growth, controlled deterioration, complex stressing vigour, and low temperature/osmotic stress tests) for prepared lots of Pinus radiata seeds were investigated in this study. The best tests were the controlled deterioration test with two days aging treatment (CD2d) test), the prechilled seedling growth test (SG+pr test), and the complex stressing vigour test (CSV test). These were then further investigated to evaluate their ability to predict the performance of different seed lots at the Forest Research Institute (FRI) nursery, Rotorua. The CD2d, SG + pr and CSV tests showed good correlation, especially with percentage of plan table seedlings at the FRI nursery. In addition, these tests seem to have met most of the AOSA's (1983) criteria for a practical vigour testing, as they are simple and can be done in a relatively short period of time. For application purposes, it is suggested that the test parameters which gave the highest correlation coefficient value with percentage of plan table seedlings in the nursery should be used as a reliable measurement. Therefore, percentage normal seedlings should be used in either the CD2d or the CSV test, whereas T50 radicle emergence seems more predictive in the SG+pr test. For application in other nurseries, these tests may still be valid, especially if pre-sowing treatment and nursery conditions are about the same as in the FRI nursery. If conditions do differ, however, the CD2d and SG + pr tests are more likely to be useful than the CSV test. This hypothesis is based on the fact that the CD2d and SG + pr tests also gave good correlations with the glasshouse (optimum conditions) and winter field tests (sub-optimum conditions). In contrast, there was no significant correlation given by the CSV test in relation to the glasshouse and winter field tests. Seed weight had a significant effect on seedling dry weight and Tso radicle emergence if there was a large seed weight variation between seed lots. In this case, generally heavier seeds had better performance than the lighter ones. If there was only small variation in overall seed weight among seed lots, however, the important effects of individual differences in seed weights were masked. The direction of further studies would seem to be to evaluate the reproducibility of correlation coefficient values and regression equations by the CD2d, SG + pr and CSV tests in the same nursery site over several sowings. Additionally, vigour test evaluation using seed lots from individual clones would also seem to be important.
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    An evaluation of osmotic pre-sowing seed treatments as a potential method for improving the germination performance of Pinus radiata D. Don seeds : a thesis presented in partial fulfilment of the requirements for the degree of Master of Agricultural Science in Seed Technology at Massey University
    (Massey University, 1992) Kusmintardjo
    This study was conducted lo characterise optimum conditions for osmotic pre-sowing treatment as an effective means of improving the germination and/or emergence performance of Pinus radiata from different seed grades. The results indicated that osmotic treatment could reduce the germination and/or emergence times of Pinus radiata seeds by 40% of controls, if treated seeds were not subsequently dried back to original moisture contents. Osmotic treatment did not alter both the uniformity and final percentage germination. Rapid germination at this rate was only obtained if seeds were treated in optimum treatment conditions, i.e. with a -1.0 MPa solution for 10 d at 20° C. The correct choice of water potential and treatment duration is crucial in determining the level of treatment benefits. At high water potential, seeds were lost due to pre-germination during treatment, while at low water potential treatment benefits were less. Treatment with sail solutions (KNO3 + KH2PO4) was better than with polyethylene glycol, an effect which seemed to be a result of differing seed moisture content attained during treatment as no pre-germination occurred during PEG treatment while the moisture content of PEG-treated seeds attained following drying was less than that attained by salt-treated seeds, Since seeds are kept in the imbibed state during treatment, the prevention of microbial proliferation is of prime importance. The use of Thiram at 1% seed weight and applied before osmotic treatment gave good protection against microbial attacks without losing treatment benefits in terms of rapid germination. Application of Thiram beyond its optimum rate should be avoided as it can delay seed germination. Treated seed should not be dried back to low moisture contents rapidly, even at ambient temperatures (22-27° C, 50-6-% RH) as drying for 4 d in these conditions resulted in a complete loss of treatment benefit. However, slow drying of osmotically treated seeds at high relative humidity (20°C, 80-85% RH) prevented the adverse effects of desiccation on germination performance. Seed dried back in this way had 30% less in median germination times relative to untreated controls. The response of osmotic treatment applied to different seed grades gave consistent results. Rapid germination due to osmotic treatment occurred in all seed grades at similar rates and was reflected in a significant increase in seedling dry weight. As the increases in seedling dry weight were more evident in larger or heavier seeds than in smaller or lighter seeds, it is suggested that osmotic treatment seems to influence relative growth at this stage of seedling development. Osmotic treatment reduced the storability of seed, although applications of this treatment after storage restored the level of vigour of aged seeds which had just begun lo decline. Although total dehydrogenase activity in osmotically treated seeds was higher than in untreated controls, there was no difference in oxygen uptake between treated and untreated controls prior to radicle emergence. It was suggested that factors other than energy production are perhaps responsible for ensuring rapid germination of treated seeds. The commercial implications of this study are potentially good. Osmotic treatment in tree seeds is no longer restricted lo using only polyethylene glycol. Improved seedling growth as a result of early emergence can help low vigour /moderate vigour seedlings become more vigorous and meet standard specifications required for outplanting.
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    Structural studies of a fucogalactoxyloglucan from pinus radiata primary cell walls : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Biochemistry at Massey University
    (Massey University, 1980) Little, John William Lester
    1. The changes in carbohydrate composition of elongating Pinus radiata primary cell walls were investigated. In the hemicellulose B extracts, a large increase in the percentage of non-starch, non-cellulosic, glucose was found to occur on cessation of cell-wall elongation. 2. By fractionation of the hemicellulose B extracts, with a variety of methods involving precipitation from an aqueous solution, a xyloglucan was purified. This xyloglucan was the major hemicellulose of the Pinus radiata hypocotyl cell wall. 3. Characterisation studies on the xyloglucan involved: quantitative analysis of the monosaccharides derived by nitric acid/urea hydrolysis; identification of the partial hydrolysis products derived by trifluoroacetic acid hydrolysis; quantitation of the sugar linkages using methylation by the Hakomori method; and analysis of the anomeric configuration of component sugars using chromium trioxide oxidation. 4. From the results a tentative structure has been suggested for the xyloglucan, consisting of a backbone of B-D-gluco-pyranose residues linked together by 1-4 glycosidic bonds, and with sidechains of single xylose residues linked through C-6 of the glucose units. Galacto and fuco-1,2- galacto sidechains are attached to some of the xylose residues, probably through the C-2 of the xylose.
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    Evaluation of the 1986-1987 radiata pine clonal trials at Forest Research, New Zealand : a thesis presented in partial fulfilment of the requirements for the degree of Master in Applied Science at Massey University
    (Massey University, 1998) Concheyro, Silvia Claudia
    Clonal forestry, the establishment of plantations using tested clones, is highly sought after by the forestry industry in New Zealand and worldwide. Clonal testing is a vital element in the process leading to clonal forestry. Two clonal trials established in 1986 and 1987 by the Forest Research Institute with juvenile ortet material have been analysed in this study. The mating design in the 1986 clones-in-family trial was single-pair crossing with amplification of the clones by fascicle cuttings. It was replicated over two sites, and the trait analysed was diameter at 1.40 m height at ages 4,7, and 10 years. The estimation of additive, non-additive and genetic variances showed a high proportion of non-additive variance compared with the additive variance at one of the sites, whereas the proportion was less important at the other site. The high non-additive component of variance can be due to important dominance or epistasis, or to C-effects confounded with the non-additive variance. This trend was similar for all three ages. Realised genetic gains were obtained from selection of clones at age 10 years for clonal deployment and breeding. For clonal deployment, realised gains were high at both sites (13% and 16%). The gains were similar at both sites provided selection was based on performance values at the site, and not on indirect selection on performance of clones at the other site. Realised gains for selection at age 10 based on the performance of clones on combined sites (10% and 13%) were less than the maximum gain obtained at each individual site. Gains based on information from both sites (10% and 12% at respective sites) were more stable than those selections at any one site. For breeding, the level of gain was significantly inferior than for clonal deployment (4% and 8%), especially when the number of clones per family was restricted to one (2% and 4%). Realised gain on combined-site selection yielded less gain than direct selection at the optimum site for selection (1% and 2%). The presence of genotype x environment interaction emphasised the need to test clones in several sites if stability of performance is desired. It is possible to obtain gain from selections made at an early age, but selections made for breeding at the age of final assessment yielded greater expected total gain and gain per unit time. The mating design in the 1987 clones-in-family trial was a 3 x 3 disconnected factorial. The trial was established on a single site and the trait analysed was percentage of Dothistroma needle infection at ages 3,4 and 7years. The mating design allowed estimation of additive, dominance and epistasis variances, which were overestimated for the lack of replication over sites. In this trial measured for Dothistroma resistance, the additive variance was the major component of the genetic variance at both ages. The evolution of components of genetic variance was confounded with the level of Dothistroma infection. The analysis of these trials indicated the need to improve the mating and field designs to improve the accuracy in the estimation of genetic parameters, highlights the importance of annual or biennual measurements to determine trends of those parameters over time, and showed the difference in gains obtained from selection for breeding and clonal deployment for early selection and selection at the age of final assessment. Accuracy in the estimation of genetic parameters can be achieved using factorial mating designs together with serial propagation to reduce the incidence of C effects, and with replication over several sites. Further considerations have to be made to find the most appropriate field and statistical design, but alpha designs are a possibility to explore. Investment in a series of carefully planned clonal trials is fundamental to the future of clonal forestry in radiata pine.
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    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.
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    Confirmation of the presence of a dothistromin biosynthetic gene cluster in the fungal forest pathogen Dothistroma pini : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Genetics at Massey University
    (Massey University, 2001) Seconi, Janet Margaret
    The polyketide dothistromin is a toxin produced by the fungus Dothistroma pini and is thought to play a role in causing Dothistroma needle blight on the pine Pinus radiata. Dothistromin is structurally similar to aflatoxin B1 (AF), a highly carcinogenic toxin with no known function, that is produced by the fungus Aspergillus parasiticus. The structural similarities between AF and dothistromin suggest that genes homologous to AF biosynthetic genes found in D. pini are dothistromin biosynthetic genes. AF biosynthetic genes in A. parasiticus and A. flavus are clustered, as are the biosynthetic genes of the structurally similar sterigmatocystin in A. nidulans. Dothistromin biosynthetic genes are also likely to be clustered. Two λ clones, λCGV1 and λBMKSA, containing different portions of this putative dothistromin cluster, have been isolated in previous studies. In this study one gene contained on the clone λCGV1 coding for a putative dothistromin ketoreductase dotA (80.2% identical to A. parasiticus AF biosynthetic gene ver-1) was disrupted with the hygromycin B resistance gene (hph) using targeted disruption via homologous recombination. dotA- mutants were tested for dothistromin production and shown to produce at least 10 - 43 times less than the wild type strain. This confirmed that dotA is involved in dothistromin biosynthesis. Further more, dotA- mutants accumulated the intermediate versicolorin A. This finding provides evidence that λCGV1 contains a portion of the dothistromin biosynthetic gene cluster and the presence of versicolorin A suggests pathway by which dothistromin is synthesised. Other genes homologous to AF and ST biosynthetic genes contained on λCGV1 can now be disrupted in order to determine the extent of the dothistromin biosynthetic cluster on λCGV1. Dothistromin deficient mutants can also be used to determine the role of dothistromin in the pathogenicity of D. pini. Further nucleotide sequencing of the clone λBMKSA revealed the promoter region and the N terminal amino acid encoding sequence of the putative dothistromin polyketide synthase PKSDOT. The partial PKSDOT sequence (amino acids 1-1426) is 62% identical to the A. parasiticus PKSA involved in AF biosynthesis. Preparations to disrupt PKSDOT were made and disruption will confirm its presence on the dothistromin biosynthetic pathway. Sequencing of λBMKSA in this study also revealed a putative dothistromin p450 monooxygenase gene, dcm1, providing more evidence that λBMKSA contains part of the dothistromin pathway. The amino acid sequence of dcml is 59% identical to CYPX from the A. parasiticus AF cluster and 56% identical to STCB from the A. nidulans ST cluster. The function of these homologs has not been ascertained. The discovery of a homolog in D. pini (a species only thought to contain genes for the first part of the AF/ST pathway) provides information about the function of these homologs in AF and ST biosynthesis.