Ten polymorphic microsatellite loci identified from a small insert genomic library for Peronospora tabacina.

Ten polymorphic microsatellite loci for the obligate biotrophic, oomycete pathogen of tobacco, Peronospora tabacina, were identified from a small insert genomic library enriched for GT motifs. Eighty-five percent of the 162 loci identified were composed of dinucleotide repeats, whereas only 4% and 11% were tri-and tetra-nucleotide repeats respectively. About 82% of all the microsatellites were perfect and within the library; only about 7% of the loci were duplicated. Primers were designed for 63 loci; 10 loci were polymorphic, 19 were monomorphic and 34 either failed to amplify or produced ambiguous/inconsistent results. The 10 polymorphic loci were characterized with 44 isolates of P. tabacina collected from tobacco plants growing in Europe, the Near East and North and South America. The number of alleles per locus was either three or four with a mean of 3.2, and the mean number of genotypes per locus was 3.6. Observed heterozygosity was 0.32-0.95, whereas expected heterozygosity was 0.44-0.69 for these loci. All loci except PT054 did not conform to the Hardy-Weinberg distribution. Polymorphic information content (PIC) for the loci was 0.35-0.69 with a mean of 0.50. These microsatellite loci provide a set of markers sufficient to perform genetic diversity and population studies of P. tabacina, and possibly other species of Peronospora.

Multiple evolutionary origins of sequestrate species in the agaricoid genus Chlorophyllum.

Chlorophyllum accommodates lamellate agaricoid species as well as sequestrate angiocarpic taxa, which do not form a monophyletic lineage within the genus. To clarify phylogenetic affinities and delimit species boundaries among sequestrate representatives of the genus, we analyzed historical and contemporary material from a broad geographic range, encompassing North America, southern Africa, eastern Asia, the Greek and Iberian peninsulas, and the Mediterranean islands of Cyprus and Lesvos. Six sequestrate lineages of Chlorophyllum were identified, which appear to have evolved in at least three independent gasteromycetation events. Multigene analysis of the nuc rDNA internal transcribed spacer region ITS1-5.8S-ITS2 (ITS) and portions of the nuc 28S rDNA (28S), RNA polymerase II second largest subunit (rpb2), and translation elongation factor 1-α (tef1) genes revealed the presence of a previously undescribed species, introduced here as Chlorophyllum levantinum, sp. nov. Its sister species C. lusitanicum, previously known only from Spain, is shown to have a wider distribution throughout the Mediterranean basin. A South African collection of Secotium gueinzii, a poorly known taxon described in 1840 and seldom appearing in literature since, was also shown to nest within Chlorophyllum. An epitype for this rare species is designated, Secotium becomes a priority synonym of Chlorophyllum, and nomenclatural implications are discussed. The phylogenetic placement of C. arizonicum is confirmed after successful sequencing of the century-old holotype and an undescribed sister lineage of this species detected. Emended descriptions of sections Chlorophyllum, Endoptychorum, and Sphaerospororum are provided to reflect current results, along with updated descriptions and extensive imagery for all known sequestrate taxa of Chlorophyllum.

Identity of the downy mildew pathogens of basil, coleus, and sage with implications for quarantine measures.

The downy mildew pathogen of basil (Ocimum spp.) has caused considerable damage throughout the past five years, and an end to the epidemics is not in sight. The downy mildew of coleus (Solenostemon spp.) is just emerging and here we report that it was very recently introduced into Germany. Although it has been recognised that these pathogens are a major threat, the identity of the pathogens is still unresolved, and so it is difficult to devise quarantine measures against them. Using morphological comparison and molecular phylogenetic reconstructions we confirmed in this study that the downy mildews of basil and coleus are unrelated to Peronospora lamii, which is a common pathogen of the weed Lamium purpureum. In addition, we conclude by the investigation of the type specimen of P. swingleii and downy mildew specimens on Salvia officinalis that the newly occurring pathogens are not identical to P. swingleii on Salvia reflexa. The taxonomy of the downy mildew pathogens of hosts from the Lamiaceae and, in particular, from the tribes Mentheae and Elsholtzieae, is discussed, and a new species is described to accommodate the downy mildew pathogen of basil and coleus, which is the first downy mildew pathogen known to be parasitic to hosts of the tribe Ocimeae.

Baobabopsis, a new genus of graminicolous downy mildews from tropical Australia, with an updated key to the genera of downy mildews.

So far 19 genera of downy mildews have been described, of which seven are parasitic to grasses. Here, we introduce a new genus, Baobabopsis, to accommodate two distinctive downy mildews, B. donbarrettii sp. nov., collected on Perotis rara in northern Australia, and B. enneapogonis sp. nov., collected on Enneapogon spp. in western and central Australia. Baobabopsis donbarrettii produced both oospores and sporangiospores that are morphologically distinct from other downy mildews on grasses. Molecular phylogenetic analyses showed that the two species of Baobabopsis occupied an isolated position among the known genera of graminicolous downy mildews. The importance of the Poaceae for the evolution of downy mildews is highlighted by the observation that more than a third of the known genera of downy mildews occur on grasses, while more than 90 % of the known species of downy mildews infect eudicots.

Towards a universal barcode of oomycetes--a comparison of the cox1 and cox2 loci.

Oomycetes are a diverse group of eukaryotes in terrestrial, limnic and marine habitats worldwide and include several devastating plant pathogens, for example Phytophthora infestans (potato late blight). The cytochrome c oxidase subunit 2 gene (cox2) has been widely used for identification, taxonomy and phylogeny of various oomycete groups. However, recently the cox1 gene was proposed as a DNA barcode marker instead, together with ITS rDNA. The cox1 locus has been used in some studies of Pythium and Phytophthora, but has rarely been used for other oomycetes, as amplification success of cox1 varies with different lineages and sample ages. To determine which out of cox1 or cox2 is best suited as a universal oomycete barcode, we compared these two genes in terms of (i) PCR efficiency for 31 representative genera, as well as for historic herbarium specimens, and (ii) sequence polymorphism, intra- and interspecific divergence. The primer sets for cox2 successfully amplified all oomycete genera tested, while cox1 failed to amplify three genera. In addition, cox2 exhibited higher PCR efficiency for historic herbarium specimens, providing easier access to barcoding-type material. Sequence data for several historic type specimens exist for cox2, but there are none for cox1. In addition, cox2 yielded higher species identification success, with higher interspecific and lower intraspecific divergences than cox1. Therefore, cox2 is suggested as a partner DNA barcode along with ITS rDNA instead of cox1. The cox2-1 spacer could be a useful marker below species level. Improved protocols and universal primers are presented for all genes to facilitate future barcoding efforts.

Evidence for high degrees of specialisation, evolutionary diversity, and morphological distinctiveness in the genus Bremia.

Bremia lactucae is one of the most important pathogens in lettuce production. Recent molecular studies revealed considerable genetic variation in this species complex. However, only few accessions from the same host have been examined for most species and no study investigating the morphological distinctiveness of phylogenetic lineages of Bremia has so far been reported. Thus it is believed that morphological species delimitation in Bremia is not feasible. In the present study, multiple accessions of neglected species, which had been described decades ago, but have not been widely accepted, were investigated, considering both multi-gene phylogenies and morphological characters. All previously described species from host genera other than Lactuca investigated, Bremia microspora, Bremia ovata, Bremia saussureae, and Bremia sonchicola, could be confirmed as distinct, host-specific entities. Also, morphological characteristics of their conidiophores and conidia allowed delimitation of these species. Therefore, not only the wide species concept to merge all Bremia species on the Asteraceae under B. lactucae is inappropriate but also their delimitation on the basis of morphological characters seems feasible. In addition, it has been shown that Bremia elliptica is phylogenetically distinct from the other species infecting the genus Lactuca, B. lactucae. It is therefore concluded that B. lactucae is most likely limited to Lactuca sativa and closely-related species, and that most species of Bremia are highly host specific. This finding might stimulate the search for durable resistance genes in genera closely related to the genus Lactuca and in divergent species of the genus itself.

The molecular phylogeny of the white blister rust genus Pustula reveals a case of underestimated biodiversity with several undescribed species on ornamentals and crop plants.

Despite their economic importance, the knowledge of the biodiversity of many plant pathogens is still fragmentary. In this study we show that this is true also for the white blister rust genus Pustula that is parasitic on several genera in the asterids, including sunflower and the gentian, Eustoma. It is revealed that several distinct species exist in Pustula, suggesting that species are mostly host genus specific. No geographic patterns were observed in the occurrence of Pustula, the host range of which includes the Araliaceae, Asteraceae, Gentianaceae, and Goodeniaceae. Evidence points to these becoming hosts as a result of jumps from the Asteraceae, with subsequent host-specific adaptation and speciation. Among the undescribed species are pathogens of economic importance, e.g. the white blister rusts of sunflower, or with still restricted geographical ranges, e.g. Pustula centaurii, which could potentially spread with international seed trade, if no quarantine restrictions are implemented.

The inclusion of downy mildews in a multi-locus-dataset and its reanalysis reveals a high degree of paraphyly in Phytophthora.

Pathogens belonging to the Oomycota, a group of heterokont, fungal-like organisms, are amongst the most notorious pathogens in agriculture. In particular, the obligate biotrophic downy mildews and the hemibiotrophic members of the genus Phytophthora are responsible for a huge variety of destructive diseases, including sudden oak death caused by P. ramorum, potato late blight caused by P. infestans, cucurbit downy mildew caused by Pseudoperonospora cubensis, and grape downy mildew caused by Plasmopara viticola. About 800 species of downy mildews and roughly 100 species of Phytophthora are currently accepted, and recent studies have revealed that these groups are closely related. However, the degree to which Phytophthora is paraphyletic and where exactly the downy mildews insert into this genus in relation to other clades could not be inferred with certainty to date. Here we present a molecular phylogeny encompassing all clades of Phytophthora as represented in a multi-locus dataset and two representatives of the monophyletic downy mildews from divergent genera. Our results demonstrate that Phytophthora is at least six times paraphyletic with respect to the downy mildews. The downy mildew representatives are consistently nested within clade 4 (contains Phytophthora palmivora), which is placed sister to clade 1 (contains Phytophthora infestans). This finding would either necessitate placing all downy mildews and Phytopthora species in a single genus, either under the oldest generic name Peronospora or by conservation the later name Phytophthora, or the description of at least six new genera within Phytophthora. The complications of both options are discussed, and it is concluded that the latter is preferable, as it warrants fewer name changes and is more practical.

Amplification of cox2 (approximately 620 bp) from 2 mg of up to 129 years old herbarium specimens, comparing 19 extraction methods and 15 polymerases.

During the past years an increasing number of studies have focussed on the use of herbarium specimens for molecular phylogenetic investigations and several comparative studies have been published. However, in the studies reported so far usually rather large amounts of material (typically around 100 mg) were sampled for DNA extraction. This equals an amount roughly equivalent to 8 cm(2) of a medium thick leaf. For investigating the phylogeny of plant pathogens, such large amounts of tissue are usually not available or would irretrievably damage the specimens. Through systematic comparison of 19 DNA extraction protocols applied to only 2 mg of infected leaf tissue and testing 15 different DNA polymerases, we could successfully amplify a mitochondrial DNA region (cox2; approximately 620 bp) from herbarium specimens well over a hundred years old. We conclude that DNA extraction and the choice of DNA polymerase are crucial factors for successful PCR amplification from small samples of historic herbarium specimens. Through a combination of suitable DNA extraction protocols and DNA polymerases, only a fraction of the preserved plant material commonly used is necessary for successful PCR amplification. This facilitates the potential use of a far larger number of preserved specimens for molecular phylogenetic investigation and provides access to a wealth of genetic information in preserved in specimens deposited in herbaria around the world without reducing their scientific or historical value.

Phylogenetic relationships of graminicolous downy mildews based on cox2 sequence data.

Graminicolous downy mildews (GDM) are an understudied, yet economically important, group of plant pathogens, which are one of the major constraints to poaceous crops in the tropics and subtropics. Here we present a first molecular phylogeny based on cox2 sequences comprising all genera of the GDM currently accepted, with both lasting (Graminivora, Poakatesthia, and Viennotia) and evanescent (Peronosclerospora, Sclerophthora, and Sclerospora) sporangiophores. In addition, all other downy mildew genera currently accepted, as well as a representative sample of other oomycete taxa, have been included. It was shown that all genera of the GDM have had a long, independent evolutionary history, and that the delineation between Peronosclerospora and Sclerospora is correct. Sclerophthora was found to be a particularly divergent taxon nested within a paraphyletic Phytophthora, but without support. The results confirm that the placement of Peronosclerospora and Sclerospora in the Saprolegniomycetidae is incorrect. Sclerophthora is not closely related to Pachymetra of the family Verrucalvaceae, and also does not belong to the Saprolegniomycetidae, but shows close affinities to the Peronosporaceae. In addition, all GDM are interspersed throughout the Peronosporaceae s lat., suggesting that a separate family for the Sclerosporaceae might not be justified.

Evolution of host resistance in a toxin-producing bacterial-fungal alliance.

The rice seedling blight fungus Rhizopus microsporus harbors endosymbiotic Burkholderia sp. for the production of the virulence factor, the antimitotic agent rhizoxin. Since the toxin highly efficiently blocks mitosis in most eukaryotes, it remained elusive how self-resistance emerged in the fungal host. In this study, rhizoxin sensitivity was systematically correlated with the nature of beta-tubulin sequences in the kingdom Fungi. A total of 49 new beta-tubulin sequences were generated for representative species of Ascomycota, Basidiomycota and Zygomycota. Rhizoxin sensitivity assays revealed two further amino acids at position 100 (Ser-100 and Ala-100), in addition to the known Ile-100 and Val-100, which convey rhizoxin resistance. All sensitive strains feature Asn-100. This hot spot was verified by modeling studies, which support the finding that rhizoxin preferentially interacts with the tubulin molecule in a cavity near position 100. Ancestral character state reconstructions conducted in a Bayesian framework suggest that rhizoxin sensitivity represents the ancestral character state in fungi, and that evolution of rhizoxin resistance took place in the ancestor of extant resistant Zygomycota. These findings support a model according to which endosymbiosis became possible through a parasitism--mutualism shift in insensitive fungi.