Tara L. Rintoul

DNA barcoding of oomycetes with cytochrome c oxidase subunit I and internal transcribed spacer

Oomycete species occupy many different environments and many ecological niches. The genera Phytophthora and Pythium for example, contain many plant pathogens which cause enormous damage to a wide range of plant species. Proper identification to the species level is a critical first step in any investigation of oomycetes, whether it is research driven or compelled by the need for rapid and accurate diagnostics during a pathogen outbreak. The use of DNA for oomycete species identification is well established, but DNA barcoding with cytochrome c oxidase subunit I (COI) is a relatively new approach that has yet to be assessed over a significant sample of oomycete genera. In this study we have sequenced COI, from 1205 isolates representing 23 genera. A comparison to internal transcribed spacer (ITS) sequences from the same isolates showed that COI identification is a practical option; complementary because it uses the mitochondrial genome instead of nuclear DNA. In some cases COI was more discriminative than ITS at the species level. This is in contrast to the large ribosomal subunit, which showed poor species resolution when sequenced from a subset of the isolates used in this study. The results described in this paper indicate that COI sequencing and the dataset generated are a valuable addition to the currently available oomycete taxonomy resources, and that both COI, the default DNA barcode supported by GenBank, and ITS, the de facto barcode accepted by the oomycete and mycology community, are acceptable and complementary DNA barcodes to be used for identification of oomycetes.

One stop shop: backbones trees for important phytopathogenic genera: I (2014)

Many fungi are pathogenic on plants and cause significant damage in agriculture and forestry. They are also part of the natural ecosystem and may play a role in regulating plant numbers/density. Morphological identification and analysis of plant pathogenic fungi, while important, is often hampered by the scarcity of discriminatory taxonomic characters and the endophytic or inconspicuous nature of these fungi. Molecular (DNA sequence) data for plant pathogenic fungi have emerged as key information for diagnostic and classification studies, although hampered in part by non-standard laboratory practices and analytical methods. To facilitate current and future research, this study provides phylogenetic synopses for 25 groups of plant pathogenic fungi in the Ascomycota, Basidiomycota, Mucormycotina (Fungi), and Oomycota, using recent molecular data, up-to-date names, and the latest taxonomic insights. Lineage-specific laboratory protocols together with advice on their application, as well as general observations, are also provided. We hope to maintain updated backbone trees of these fungal lineages over time and to publish them jointly as new data emerge. Researchers of plant pathogenic fungi not covered by the present study are invited to join this future effort. Bipolaris, Botryosphaeriaceae, Botryosphaeria, Botrytis, Choanephora, Colletotrichum, Curvularia, Diaporthe, Diplodia, Dothiorella, Fusarium, Gilbertella, Lasiodiplodia, Mucor, Neofusicoccum, Pestalotiopsis, Phyllosticta, Phytophthora, Puccinia, Pyrenophora, Pythium, Rhizopus, Stagonosporopsis, Ustilago and Verticillium are dealt with in this paper.

Fungal Planet description sheets: 371-399

Novel species of fungi described in the present study include the following from Australia: Neoseptorioides eucalypti gen. & sp. nov. from Eucalyptus radiata leaves, Phytophthora gondwanensis from soil, Diaporthe tulliensis from rotted stem ends of Theobroma cacao fruit, Diaporthe vawdreyi from fruit rot of Psidium guajava, Magnaporthiopsis agrostidis from rotted roots of Agrostis stolonifera and Semifissispora natalis from Eucalyptus leaf litter. Furthermore, Neopestalotiopsis egyptiaca is described from Mangifera indica leaves (Egypt), Roussoella mexicana from Coffea arabica leaves (Mexico), Calonectria monticola from soil (Thailand), Hygrocybe jackmanii from littoral sand dunes (Canada), Lindgomyces madisonensis from submerged decorticated wood (USA), Neofabraea brasiliensis from Malus domestica (Brazil), Geastrum diosiae from litter (Argentina), Ganoderma wiiroense on angiosperms (Ghana), Arthrinium gutiae from the gut of a grasshopper (India), Pyrenochaeta telephoni from the screen of a mobile phone (India) and Xenoleptographium phialoconidium gen. & sp. nov. on exposed xylem tissues of Gmelina arborea (Indonesia). Several novelties are introduced from Spain, namely Psathyrella complutensis on loamy soil, Chlorophyllum lusitanicum on nitrified grasslands (incl. Chlorophyllum arizonicum comb. nov.), Aspergillus citocrescens from cave sediment and Lotinia verna gen. & sp. nov. from muddy soil. Novel foliicolous taxa from South Africa include Phyllosticta carissicola from Carissa macrocarpa, Pseudopyricularia hagahagae from Cyperaceae and Zeloasperisporium searsiae from Searsia chirindensis. Furthermore, Neophaeococcomyces is introduced as a novel genus, with two new combinations, N. aloes and N. catenatus. Several foliicolous novelties are recorded from La Réunion, France, namely Ochroconis pandanicola from Pandanus utilis, Neosulcatispora agaves gen. & sp. nov. from Agave vera-cruz, Pilidium eucalyptorum from Eucalyptus robusta, Strelitziana syzygii from Syzygium jambos (incl. Strelitzianaceae fam. nov.) and Pseudobeltrania ocoteae from Ocotea obtusata (Beltraniaceae emend.). Morphological and culture characteristics along with ITS DNA barcodes are provided for all taxa.

Phytopythium: molecular phylogeny and systematics.

The genus Phytopythium (Peronosporales) has been described, but a complete circumscription has not yet been presented. In the present paper we provide molecular-based evidence that members of Pythium clade K as described by Lévesque & de Cock (2004) belong to Phytopythium. Maximum likelihood and Bayesian phylogenetic analysis of the nuclear ribosomal DNA (LSU and SSU) and mitochondrial DNA cytochrome oxidase subunit 1 (COI) as well as statistical analyses of pairwise distances strongly support the status of Phytopythium as a separate phylogenetic entity. Phytopythium is morphologically intermediate between the genera Phytophthora and Pythium. It is unique in having papillate, internally proliferating sporangia and cylindrical or lobate antheridia. The formal transfer of clade K species to Phytopythium and a comparison with morphologically similar species of the genera Pythium and Phytophthora is presented. A new species is described, Phytopythium mirpurense.

Seasonality and distribution of epilithic diatoms, macroalgae and macrophytes in a spring-fed stream system in Ontario, Canada

A study of the epilithic diatom, macroalgal and macrophyte communities from a spring-fed stream in Ontario, Canada was undertaken from September 1996 to July 1997. The relative abundance of the epilithic diatom flora, percent cover of macroalgal and macrophyte taxa, and several physical and chemical stream conditions were monitored along a 20-m stretch at each of four sites, approximately every 2 months. Several stream conditions were relatively constant over the sampling period (pH, maximum width and maximum depth), while others exhibited a distinct seasonal pattern (water temperature, specific conductance and daylength) and some fluctuated strongly with no discernable seasonal pattern (turbidity, current velocity). A total of 124 taxa were identified from the four sites, including 79 epilithic diatoms, three macroalgal diatom species (large gelatinous masses), one cyanobacterium, two red algae, eight green algae, one chrysophyte alga, one tribophyte alga, three mosses, three horsetails and 23 angiosperm taxa. Species richness was positively correlated to stream channel maximum width and depth, indicating that the total number of species tends to increase in a downstream direction. Distribution of several diatom and macroalgal species was significantly correlated to stream conditions (e.g. Gomphonema parvulum and Phormidium subfuscum with current velocity); however, the vast majority of species did not display seasonal variation in abundance that could be explained by changes in stream conditions. Many of the taxa identified from Blue Springs Creek are common elsewhere in North America.

Coloring genetically modified soybean grains with anthocyanins by suppression of the proanthocyanidin genes ANR1 and ANR2

Detection and quantification of the levels of adventitious presence of genetically modified (GM) soybeans in non-GM grain shipments currently requires sophisticated tests that can have issues with their reproducibility. We show here that pigment biosynthesis in the soybean seed coat can be manipulated to provide a distinct color that would enable the simple visible detection of the GM soybean grain. We observed that a distinct red-brown grain color could be engineered by the simultaneous suppression of two proanthocyanidin (PA) genes, ANTHOCYANIDINREDUCTASE1 (ANR1) and ANR2. Multiple reaction monitoring by liquid chromatography tandem mass spectrometry was used to quantify differentially accumulated seed coat metabolites, and revealed the redirection of metabolic flux into the anthocyanin pigment pathway and unexpectedly the flavonol-3-O-glucoside pathway. The upregulations of anthocyanin isogenes (DFR1 and GST26) and the anthocyanin/flavonol-3-O-glycosyltransferase (UGT78K2) were identified by quantitative RT-PCR to be endogenous feedback and feedforward responses to overaccumulation of upstream flavonoid intermediates resulting from ANR1 and ANR2 suppressions. These results suggested the transcription of flavonoid genes to be a key component of the mechanism responsible for the redirection of metabolite flux. This report identifies the suppression of PA genes to be a novel approach for engineering pigmentation in soybean grains.

Multigene phylogenetic analyses to delimit new species in fungal plant pathogens.

Supporting the identification of unknown strains or specimens by sequencing a genetic marker commonly used for phylogenetics or DNA barcoding is now standard practice for mycologists and plant pathologists. Does one have a new species when a strain differs by a few base pairs when compared to reference sequences from taxonomically well-characterized species that do not differ morphologically from this new strain? If variation at the intra- and interspecific levels for the locus used for identification is already understood for all the closely related species, it is possible to make a reliable prediction of a new species status, but ultimately this question can only be properly addressed by determining the presence or absence of gene flow among a group of strains of the putative new species and strains of previously delimited species. The Phylogenetic Species Concept (PSC) and its assessment using multigene phylogeny and Genealogical Concordance Phylogenetic Species Recognition (GCPSR) are the basis for this chapter. The theoretical framework and a variety of tools to apply these concepts are explained, to assist in the assessment of whether a species is distinct or new when confronted with some sequence divergence from reference data.

Soybean seeds overexpressing asparaginase exhibit reduced nitrogen concentration

In soybean seed, a correlation has been observed between the concentration of free asparagine at mid-maturation and protein concentration at maturity. In this study, a Phaseolus vulgaris K+ -dependent asparaginase cDNA, PvAspG2, was expressed in transgenic soybean under the control of the embryo specific promoter of the β-subunit of β-conglycinin. Three lines were isolated having high expression of the transgene at the transcript, protein and enzyme activity levels at mid-maturation, with a 20- to 40-fold higher asparaginase activity in embryo than a control line expressing β-glucuronidase. Increased asparaginase activity was associated with a reduction in free asparagine levels as a percentage of total free amino acids, by 11-18%, and an increase in free aspartic acid levels, by 25-60%. Two of the lines had reduced nitrogen concentration in mature seed as determined by nitrogen analysis, by 9-13%. Their levels of extractible globulins were reduced by 11-30%. This was accompanied by an increase in oil concentration, by 5-8%. The lack of change in nitrogen concentration in the third transgenic line was correlated with an increase in free glutamic acid levels by approximately 40% at mid-maturation.