BackgroundPhytomyxids (plasmodiophorids and phagomyxids) are cosmopolitan, obligate biotrophic protist parasites of plants, diatoms, oomycetes and brown algae. Plasmodiophorids are best known as pathogens or vectors for viruses of arable crops (e.g. clubroot in brassicas, powdery potato scab, and rhizomania in sugar beet). Some phytomyxid parasites are of considerable economic and ecologic importance globally, and their hosts include important species in marine and terrestrial environments. However most phytomyxid diversity remains uncharacterised and knowledge of their relationships with host taxa is very fragmentary.ResultsOur molecular and morphological analyses of phytomyxid isolates–including for the first time oomycete and sea-grass parasites–demonstrate two cross-kingdom host shifts between closely related parasite species: between angiosperms and oomycetes, and from diatoms/brown algae to angiosperms. Switching between such phylogenetically distant hosts is generally unknown in host-dependent eukaryote parasites. We reveal novel plasmodiophorid lineages in soils, suggesting a much higher diversity than previously known, and also present the most comprehensive phytomyxid phylogeny to date.ConclusionSuch large-scale host shifts between closely related obligate biotrophic eukaryote parasites is to our knowledge unique to phytomyxids. Phytomyxids may readily adapt to a wide diversity of new hosts because they have retained the ability to covertly infect alternative hosts. A high cryptic diversity and ubiquitous distribution in agricultural and natural habitats implies that in a changing environment phytomyxids could threaten the productivity of key species in marine and terrestrial environments alike via host shift speciation.

Background: Recent phylogenomic analyses have revolutionized our view of eukaryote evolution by revealing unexpected relationships between and within the eukaryotic supergroups. However, for several groups of uncultivable protists, only the ribosomal RNA genes and a handful of proteins are available, often leading to unresolved evolutionary relationships. A striking example concerns the supergroup Rhizaria, which comprises several groups of uncultivable free-living protists such as radiolarians, foraminiferans and gromiids, as well as the parasitic plasmodiophorids and haplosporids. Thus far, the relationships within this supergroup have been inferred almost exclusively from rRNA, actin, and polyubiquitin genes, and remain poorly resolved. To address this, we have generated large Expressed Sequence Tag (EST) datasets for 5 species of Rhizaria belonging to 3 important groups: Acantharea (Astrolonche sp., Phyllostaurus sp.), Phytomyxea (Spongospora subterranea, Plasmodiophora brassicae) and Gromiida (Gromia sphaerica).

The obligate biotrophic pathogen Plasmodiophora brassicae causes clubroot disease in Arabidopsis thaliana, which is characterized by large root galls. Salicylic acid (SA) production is a defence response in plants, and its methyl ester is involved in systemic signalling. Plasmodiophora brassicae seems to suppress plant defence reactions, but information on how this is achieved is scarce. Here, we profile the changes in SA metabolism during Arabidopsis clubroot disease. The accumulation of SA and the emission of methylated SA (methyl salicylate, MeSA) were observed in P. brassicae-infected Arabidopsis 28 days after inoculation. There is evidence that MeSA is transported from infected roots to the upper plant. Analysis of the mutant Atbsmt1, deficient in the methylation of SA, indicated that the Arabidopsis SA methyltransferase was not responsible for alterations in clubroot symptoms. We found that P. brassicae possesses a methyltransferase (PbBSMT) with homology to plant methyltransferases. The PbBSMT gene is maximally transcribed when SA production is highest. By heterologous expression and enzymatic analyses, we showed that PbBSMT can methylate SA, benzoic and anthranilic acids.

Summary• Microscopic evidence suggests that fungi forming endosymbioses with liverworts in the Marchantiales are arbuscular mycorrhizal (AM) fungi from the Glomeromycota. Polymerase chain reaction amplification of ribosomal sequences confirmed that endophytes of the New Zealand liverwort, Marchantia foliacea , were members of the genus Glomus .• Endophytes from two Glomus rDNA phylotypes were repeatedly isolated from geographically separated liverwort samples.• Multiple phylotypes were present in the same liverwort patch. The colonizing Glomus species exhibited substantial internal transcribed spacer sequence variation within phylotypes.• This work suggests that certain liverwort species may serve as a model for studying DNA sequence variation in colonizing AM phylotypes and specificity in AM-host relationships.

Molecular examination of the ribosomal internal transcribed spacer (ITS) region in potato cyst nematodes (PCN) is described. The ITS was amplified and sequenced from a number of PCN collections. A low level of sequence variation was found between Globodera rostochiensis, G. pallida, and a Peruvian PCN collection, but no variation within Australasian collections of species was noted. Polymerase chain reaction (PCR) primers based upon the G. rostochiensis-G. pallida sequence differences were designed and successfully used to identify mixed PCN species in a single PCR reaction.

The development of specific oligonucleotide primers for Plasmodiophora brassicae has led to a nested polymerase chain reaction (PCR) detection method for P. brassicae in soil and water. Initially, the PCR was used to amplify a section of the rDNA repeat. The PCR products were sequenced and the data used to design primers that were directed at the ribosomal RNA genes and internal transcribed spacer regions. Specificity was tested against more than 40 common soil organisms, host plants, and spore suspension contaminants, as well as P. brassicae isolates from around Australia and the world. Sensitivity was determined to be 0.1 fentograms (fg; 10(-15) g) for pure template and as low as 1,000 spores per g of potting mix. In soil, P. brassicae was detected in all soils where the inoculum was sufficient to result in clubroot symptoms. Also outlined is a simple method of DNA extraction from soil.

Plasmodiophora brassicae is an intracellular pathogen that infects plants in the Brassicaceae family. Although an important pathogen group, information on the genomic makeup of the plasmodiophorids is almost completely lacking. We performed suppression subtractive hybridization (SSH) between RNA from P. brassicae-infected and uninfected Arabidopsis tissue, then screened 232 clones from the resulting SSH library. In addition, we used an oligo-capping procedure to screen 305 full-length cDNA clones from the infected tissue. A total of 76 new P. brassicae gene sequences were identified, the majority of which were extended to full length at the 5' end by the use of RACE amplification. Many of the unisequences were predicted to contain signal peptides for ER translocation. Although we located few sequences in total, these markedly increase available data from the plasmodiophorids, and provide new opportunities to examine plasmodiophorid biology. Our study also points towards the best methods for future plasmodiophorid gene discovery.

The ribosomal internal transcribed spacer (ITS) was sequenced from several collections of Spongospor,l subtcrr,me,l from Europe, Peru, and Australasia. No sequence variation was detected between any of the Australasian or European collections with the exception of one from Inverness (Scotland) which was identical to the two Peruvian samples. PCR primers designed from the ITS sequence were used successfully to detectS. subterranea in DNA extracts from potato sc,1 b lesions.