Comparative Analyses of Complete Peronosporaceae (Oomycota) Mitogenome Sequences-Insights into Structural Evolution and Phylogeny.

Members of the Peronosporaceae (Oomycota, Chromista), which currently consists of 25 genera and approximately 1,000 recognized species, are responsible for disease on a wide range of plant hosts. Molecular phylogenetic analyses over the last two decades have improved our understanding of evolutionary relationships within Peronosporaceae. To date, 16 numbered and three named clades have been recognized; it is clear from these studies that the current taxonomy does not reflect evolutionary relationships. Whole organelle genome sequences are an increasingly important source of phylogenetic information, and in this study, we present comparative and phylogenetic analyses of mitogenome sequences from 15 of the 19 currently recognized clades of Peronosporaceae, including 44 newly assembled sequences. Our analyses suggest strong conservation of mitogenome size and gene content across Peronosporaceae but, as previous studies have suggested, limited conservation of synteny. Specifically, we identified 28 distinct syntenies amongst the 71 examined isolates. Moreover, 19 of the isolates contained inverted or direct repeats, suggesting repeated sequences may be more common than previously thought. In terms of phylogenetic relationships, our analyses of 34 concatenated mitochondrial gene sequences resulted in a topology that was broadly consistent with previous studies. However, unlike previous studies concatenated mitochondrial sequences provided strong support for higher-level relationships within the family.

The mitogenome of Phytophthora agathidicida: Evidence for a not so recent arrival of the "kauri killing" Phytophthora in New Zealand.

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.

Two new Nothophytophthora species from streams in Ireland and Northern Ireland: Nothophytophthora irlandica and N. lirii sp. nov.

Slow growing oomycete isolates with morphological resemblance to Phytophthora were obtained from forest streams during routine monitoring for the EU quarantine forest pathogen Phytophthora ramorum in Ireland and Northern Ireland. Internal Transcribed Spacer (ITS) sequence analysis indicated that they belonged to two previously unknown species of Nothophytophthora, a recently erected sister genus of Phytophthora. Morphological and temperature-growth studies were carried out to characterise both new species. In addition, Bayesian and Maximum-Likelihood analyses of nuclear 5-loci and mitochondrial 3-loci datasets were performed to resolve the phylogenetic positions of the two new species. Both species were sterile, formed chlamydospores and partially caducous nonpapillate sporangia, and showed slower growth than any of the six known Nothophytophthora species. In all phylogenetic analyses both species formed distinct, strongly supported clades, closely related to N. chlamydospora and N. valdiviana from Chile. Based on their unique combination of morphological and physiological characters and their distinct phylogenetic positions the two new species are described as Nothophytophthora irlandica sp. nov. and N. lirii sp. nov. Their potential lifestyle and geographic origin are discussed.

A LAMP at the end of the tunnel: A rapid, field deployable assay for the kauri dieback pathogen, Phytophthora agathidicida.

The root rot causing oomycete, Phytophthora agathidicida, threatens the long-term survival of the iconic New Zealand kauri. Currently, testing for this pathogen involves an extended soil bioassay that takes 14-20 days and requires specialised staff, consumables, and infrastructure. Here we describe a loop-mediated isothermal amplification (LAMP) assay for the detection of P. agathidicida that targets a portion of the mitochondrial apocytochrome b coding sequence. This assay has high specificity and sensitivity; it did not cross react with a range of other Phytophthora isolates and detected as little as 1 fg of total P. agathidicida DNA or 116 copies of the target locus. Assay performance was further investigated by testing plant tissue baits from flooded soil samples using both the extended soil bioassay and LAMP testing of DNA extracted from baits. In these comparisons, P. agathidicida was detected more frequently using the LAMP test. In addition to greater sensitivity, by removing the need for culturing, the hybrid baiting plus LAMP approach is more cost effective than the extended soil bioassay and, importantly, does not require a centralised laboratory facility with specialised staff, consumables, and equipment. Such testing will allow us to address outstanding questions about P. agathidicida. For example, the hybrid approach could enable monitoring of the pathogen beyond areas with visible disease symptoms, allow direct evaluation of rates and patterns of spread, and allow the effectiveness of disease control to be evaluated. The hybrid LAMP bioassay also has the potential to empower local communities to evaluate the pathogen status of local kauri stands, providing information for disease management and conservation initiatives.

The Pacific Biosciences de novo assembled genome dataset from a parthenogenetic New Zealand wild population of the longhorned tick, Haemaphysalis longicornis Neumann, 1901.

The longhorned tick, Haemaphysalis longicornis, feeds upon a wide range of bird and mammalian hosts. Mammalian hosts include cattle, deer, sheep, goats, humans, and horses. This tick is known to transmit a number of pathogens causing tick-borne diseases, and was the vector of a recent serious outbreak of oriental theileriosis in New Zealand. A New Zealand-USA consortium was established to sequence, assemble, and annotate the genome of this tick, using ticks obtained from New Zealand's North Island. In New Zealand, the tick is considered exclusively parthenogenetic and this trait was deemed useful for genome assembly. Very high molecular weight genomic DNA was sequenced on the Illumina HiSeq4000 and the long-read Pac Bio Sequel platforms. Twenty-eight SMRT cells produced a total of 21.3 million reads which were assembled with Canu on a reserved supercomputer node with access to 12 TB of RAM, running continuously for over 24 days. The final assembly dataset consisted of 34,211 contigs with an average contig length of 215,205 bp. The quality of the annotated genome was assessed by BUSCO analysis, an approach that provides quantitative measures for the quality of an assembled genome. Over 95% of the BUSCO gene set was found in the assembled genome. Only 48 of the 1066 BUSCO genes were missing and only 9 were present in a fragmented condition. The raw sequencing reads and the assembled contigs/scaffolds are archived at the National Center for Biotechnology Information.

The arbuscular mycorrhizal fungi colonising roots and root nodules of New Zealand kauri Agathis australis.

As the only endemic member in New Zealand of the ancient conifer family, Araucariaceae, Agathis australis is an ideal species to study putatively long-evolved mycorrhizal symbioses. However, little is known about A. australis root and nodular arbuscular mycorrhizal fungi (AMF), and how mycorrhizal colonisation occurs. We used light, scanning and transmission electron microscopy to characterise colonisation, and 454-sequencing to identify the AMF associated with A. australis roots and nodules. We interpreted the results in terms of the edaphic characteristics of the A. australis-influenced ecosystem. Representatives of five families of Glomeromycota were identified via high-throughput pyrosequencing. Imaging studies showed that there is abundant, but not ubiquitous, colonisation of nodules, which suggests that nodules are mostly colonised by horizontal transmission. Roots were also found to harbour AMF. This study is the first to demonstrate the multiple Glomeromycota lineages associated with A. australis including some that may not have been previously detected.

Shoot transcriptome of the giant reed, Arundo donax.

The giant reed, Arundo donax, is a perennial grass species that has become an invasive plant in many countries. Expansive stands of A. donax have significant negative impacts on available water resources and efforts are underway to identify biological control agents against this species. The giant reed grows under adverse environmental conditions, displaying insensitivity to drought stress, flooding, heavy metals, salinity and herbaceous competition, thus hampering control programs. To establish a foundational molecular dataset, we used an llumina Hi-Seq protocol to sequence the transcriptome of actively growing shoots from an invasive genotype collected along the Rio Grande River, bordering Texas and Mexico. We report the assembly of 27,491 high confidence transcripts (≥200 bp) with at least 70% coverage of known genes in other Poaceae species. Of these 13,080 (47.58%), 6165 (22.43%) and 8246 (30.0%) transcripts have sequence similarity to known, domain-containing and conserved hypothetical proteins, respectively. We also report 75,590 low confidence transcripts supported by both trans-ABBySS and Velvet-Oases de novo assembly pipelines. Within the low confidence subset of transcripts we identified partial hits to known (19,021; 25.16%), domain-containing (7093; 9.38%) and conserved hypothetical (16,647; 22.02%) proteins. Additionally 32,829 (43.43%) transcripts encode putative hypothetical proteins unique to A. donax. Functional annotation resulted in 5,550 and 6,070 transcripts with assigned Gene Ontology and KEGG pathway information, respectively. The most abundant KEGG pathways are spliceosome, ribosome, ubiquitin mediated proteolysis, plant-pathogen interaction, RNA degradation and oxidative phosphorylation metabolic pathway. Furthermore, we also found 12, 9, and 4 transcripts annotated as stress-related, heat stress, and water stress proteins, respectively. We envisage that these resources will promote and facilitate studies of the abiotic stress capabilities of this exotic plant species, which facilitates its invasive capacity.