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    The mitogenome of Phytophthora agathidicida: Evidence for a not so recent arrival of the "kauri killing" Phytophthora in New Zealand
    (PLOS, 2021-05-21) Winkworth RC; Bellgard SE; McLenachan PA; Lockhart PJ; Blair JE
    Phytophthora agathidicida is associated with a root rot that threatens the long-term survival of the iconic New Zealand kauri. Although it is widely assumed that this pathogen arrived in New Zealand post-1945, this hypothesis has yet to be formally tested. Here we describe evolutionary analyses aimed at evaluating this and two alternative hypotheses. As a basis for our analyses, we assembled complete mitochondrial genome sequences from 16 accessions representing the geographic range of P. agathidicida as well as those of five other members of Phytophthora clade 5. All 21 mitogenome sequences were very similar, differing little in size with all sharing the same gene content and arrangement. We first examined the temporal origins of genetic diversity using a pair of calibration schemes. Both resulted in similar age estimates; specifically, a mean age of 303.0-304.4 years and 95% HPDs of 206.9-414.6 years for the most recent common ancestor of the included isolates. We then used phylogenetic tree building and network analyses to investigate the geographic distribution of the genetic diversity. Four geographically distinct genetic groups were recognised within P. agathidicida. Taken together the inferred age and geographic distribution of the sampled mitogenome diversity suggests that this pathogen diversified following arrival in New Zealand several hundred to several thousand years ago. This conclusion is consistent with the emergence of kauri dieback disease being a consequence of recent changes in the relationship between the pathogen, host, and environment rather than a post-1945 introduction of the causal pathogen into New Zealand.
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    Chromosome-level assembly of the Phytophthora agathidicida genome reveals adaptation in effector gene families
    (Frontiers Media S.A., 2022-11-02) Cox MP; Guo Y; Winter DJ; Sen D; Cauldron NC; Shiller J; Bradley EL; Ganley AR; Gerth ML; Lacey RF; McDougal RL; Panda P; Williams NM; Grunwald NJ; Mesarich CH; Bradshaw RE; Hane J
    Phytophthora species are notorious plant pathogens, with some causing devastating tree diseases that threaten the survival of their host species. One such example is Phytophthora agathidicida, the causal agent of kauri dieback - a root and trunk rot disease that kills the ancient, iconic and culturally significant tree species, Agathis australis (New Zealand kauri). A deeper understanding of how Phytophthora pathogens infect their hosts and cause disease is critical for the development of effective treatments. Such an understanding can be gained by interrogating pathogen genomes for effector genes, which are involved in virulence or pathogenicity. Although genome sequencing has become more affordable, the complete assembly of Phytophthora genomes has been problematic, particularly for those with a high abundance of repetitive sequences. Therefore, effector genes located in repetitive regions could be truncated or missed in a fragmented genome assembly. Using a combination of long-read PacBio sequences, chromatin conformation capture (Hi-C) and Illumina short reads, we assembled the P. agathidicida genome into ten complete chromosomes, with a genome size of 57 Mb including 34% repeats. This is the first Phytophthora genome assembled to chromosome level and it reveals a high level of syntenic conservation with the complete genome of Peronospora effusa, the only other completely assembled genome sequence of an oomycete. All P. agathidicida chromosomes have clearly defined centromeres and contain candidate effector genes such as RXLRs and CRNs, but in different proportions, reflecting the presence of gene family clusters. Candidate effector genes are predominantly found in gene-poor, repeat-rich regions of the genome, and in some cases showed a high degree of duplication. Analysis of candidate RXLR effector genes that occur in multicopy gene families indicated half of them were not expressed in planta. Candidate CRN effector gene families showed evidence of transposon-mediated recombination leading to new combinations of protein domains, both within and between chromosomes. Further analysis of this complete genome assembly will help inform new methods of disease control against P. agathidicida and other Phytophthora species, ultimately helping decipher how Phytophthora pathogens have evolved to shape their effector repertoires and how they might adapt in the future.
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    Genetic studies of Phytophthora on Theobroma cacao from East New Britain and Bougainville (Papua New Guinea) : a thesis presented in partial fulfilment of the requirements for the degree of Master of AgriScience (Horticulture) at Massey University, Palmerston North, New Zealand
    (Massey University, 2016) Butubu, James
    DNA was extracted from 14 Phytophthora isolates from Theobroma cacao plants (SG2 hybrids, hybrid derived clones and Trinitario varieties) collected from New Britain and Bougainville in Papua New Guinea (PNG). A fragment of the mitochondrial genome cytochrome b (cytb) region was amplified from these DNAs using the polymerase chain reaction (PCR) and compared to cytochrome b sequences from Phytophthora palmivora and other Phytophthora (two isolates previously isolated from cocoa lesions and two P. palmivora isolates obtained from culture collection-Australia). All isolates were identical in their cytb gene sequence and similar to P. palmivora. Additionally, we sequenced the mitochondrial genomes of four isolates from PNG. The syntenic arrangement of genes in one complete assembly was compared with other published mitochondrial genomes. The sequences of four mitochondrial genes (COII, nad2, rps10 and SecY) from the four PNG isolates were aligned with orthologues from accessions of P. palmivora and other Phytophthora species available in the NCBI Genbank reference database. A concatenated data matrix was produced with 2,295 homologous sequence positions. 34 accessions of Phytophthora (including 14 P. palmivora) were used to construct a maximum likelihood tree of phylogenetic relationships. This reconstruction recovered all 10 major clades of Phytophthora previously reported. In this phylogenetic reconstruction, the four PNG isolates were clearly identifiable as P. palmivora and these were closely related to the Clade 4 Phytophthora species P. megakarya and P. quercetora. Of the genes analysed, COII showed greatest variability, resolving P. palmivora into three sub groups. COII was sequenced in all P. palmivora isolates from PNG and used to reconstruct an ML tree. The phylogenetic analyses suggested a potential origin for the PNG strain of P. palmivora in Samoa. Syntenic comparisons of P. palmivora and other clade 4 species identified a potential target for developing a Loop Mediated Amplification (LAMP) assay for P. palmivora near the atpH gene region. DNA amplification primers were designed for this region using PrimerExplorer, V4, Eiken Chemical CO.Ltd) and validated against available DNAs for Clade 4 and other Phytophthora species.
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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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    Development of an in vitro assay to screen Agathis australis (kauri) for resistance to Phytophthora agathidicida : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science at Massey University, Manawatū, New Zealand
    (Massey University, 2017) Herewini, Echo
    The iconic Agathis australis (kauri) of New Zealand, is under serious threat from kauri dieback disease caused by the soil-borne pathogen Phytophthora agathidicida. Infected kauri express symptoms of root and collar rot, bleeding resins at the base of the trunk, yellowing of foliage, canopy thinning, and tree mortality. Phytophthora agathidicida was first associated with kauri decline in 1972, where it was initially identified as P. heveae however, there was some uncertainty about its significance and taxonomy. The pathogen was officially identified as a new organism in 2008 and was called Phytophthora taxon Agathis until its formal description as Phytophthora agathidicida in 2015. This pathogen is easily vectored through root to root contact and mobile zoospores. Management and research has focused on mapping pathogen distribution, reducing spread, improving detection, ex situ conservation and clonal production using tissue culture techniques. In order to gain better understanding of the disease epidemiology and to develop better breeding programmes, a reliable in vitro resistance screening assay is required. This research focused on the development of a screening assay using detached leaves from tissue culture material as a means of accelerating screening assays compared to the more labour-intensive root inoculation assays. Foliar inoculations and assessment techniques were initially optimised on kauri leaves from tissue culture lines. The most successful inoculation method involved placing P. agathidicida-colonised agar plugs on wounded detached leaves. The assay was further tested on 2 year old kauri seedlings. Variation in susceptibility across kauri genotypes and leaf age, and variation in virulence among P. agathidicida isolates was observed. To further investigate the impact of leaf age on lesion extension, an assay was conducted on detached leaves from six rooted kauri saplings over 5 years of age, across three leaf age groups with P. agathidicida, P. multivora, and P. cinnamomi. Variation in virulence among these Phytophthora species was observed. Leaf necrosis was most severe with young tissue and susceptibility tended to decrease with increasing leaf age. Preliminary studies with 50 kauri clones identified different levels of susceptibility and tolerance across the different genotypes to P. agathidicida. The methods developed within this study have increased our understanding of the overall response of kauri to P. agathidicida foliar inoculations. This study demonstrated variation in the susceptability of kauri foliage to Phytophthora inoculation, although no complete resistance was observed. Further work is required to determine if there is a relationship between root and leaf responses which will help establish if in vitro genotypic variation can accurately predict natural genotypic variation seen within kauri forests.