Risk Factors for Plasmodium falciparum Gametocyte Positivity in a Longitudinal Cohort

Malaria transmission intensity is highly heterogeneous even at a very small scale. Implementing targeted intervention in malaria transmission hotspots offers the potential to reduce the burden of disease both locally and in adjacent areas. Transmission of malaria parasites from man to mosquito requires the production of gametocyte stage parasites. Cluster analysis of a 19-year long cohort study for gametocyte carriage revealed spatially defined gametocyte hotspots that occurred during the time when chloroquine was the drug used for clinical case treatment. In addition to known risk factors for gametocyte carriage, notably young age (<15 years old) and associated with a clinical episode, blood groups B and O increased risk compared to groups A and AB. A hotspot of clinical P. falciparum clinical episodes that overlapped the gametocyte hotspots was also identified. Gametocyte positivity was found to be increased in individuals who had been treated with chloroquine, as opposed to other drug treatment regimens, for a clinical P. falciparum episode up to 30 days previously. It seems likely the hotspots were generated by a vicious circle of ineffective treatment of clinical cases and concomitant gametocyte production in a sub-population characterized by an increased prevalence of all the identified risk factors. While rapid access to treatment with an effective anti-malarial can reduce the duration of gametocyte carriage and onward parasite transmission, localised hotspots represent a challenge to malaria control and eventual eradication.     

Reclassification of an enigmatic downy mildew species on lovegrass (Eragrostis) to the new genus Eraphthora, with a key to the genera of the Peronosporaceae

Graminicolous downy mildews affect economical important crops like sorghum, maize and pearl millet, and also a variety of wild grasses, which pathogens have hitherto been much less studied. This also applies to an enigmatic downy mildew from Eragrostis cilianensis, which exhibits a chimeric appearance, with similarities to Basidiophora, Sclerophthora, and Sclerospora. In this study, the phylogenetic relationship of lovegrass downy mildew with respect to other genera of the Peronosporaceae was investigated and the characteristics of genera potentially associated with lovegrass downy mildew summarised from historic studies. A key based on the morphology of asexual stages is presented for all genera of downy mildews hitherto described. Based on morphological characteristics and sequence divergence, Sclerospora butleri is transferred to a new genus, Eraphthora. Lovegrass downy mildew seems to be widely distributed, as in addition to previous records from Malawi, India and Australia, we could now sample one specimen from Europe (Spilimbergo, Italy). Currently, it is unclear if Eraphthora butleri is able to infect the crop species Eragrostis tef, which might be a potential host of economic importance.     

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.     

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.     

Cryptic diversity of <Emphasis Type="Italic">Plasmopara viticola</Emphasis> (Oomycota,Peronosporaceae) in North America

Plasmopara viticola is the causal agent of grapevine downy mildew and is among the most important diseases in viticulture. It originates from North America, where it coevolved with wild Vitis species. Beginning in the 1870s it turned into a global epidemic that has been causing severe yield losses. It is generally believed that a single species is causing downy mildew on a large variety of economically important cultivars. Here we report, based on one nuclear and two mitochondrial markers, that isolates from vineyards in the United States fall into three highly distinct phylogenetic lineages. One of these contains European strains and affects Vitis vinifera cultivars, while the other two lineages affect also other species of Vitis. The divergence between these lineages is high, and, judging from the genetic variation in other Plasmopara lineages, might reflect distinct species. Due to the potentially significant implications for quarantine regulations and resistance breeding, detailed studies will be necessary to clarify whether these genetically distinct lineages occur outside of North America or are still confined there.     

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.