Diversity of cultured isolates and field populations of the arbuscular mycorrhizal fungus "Glomus intraradices" : development and application of molecular detection methods for mitochondrial haplotypes

Today’s plant communities have evolved together with arbuscular mycorrhizal fungi (AMF, Glomeromycota) for millions of years. In “arbuscular mycorrhiza”, a mutualistic symbiosis, plants provide carbohydrates to the fungi, which in turn make mineral nutrients like phosphate or nitrogen available to the plants. AMF species diversity is generally higher in natural sites than in agroecosystems, where it can be strongly reduced. The detection of AMF is either based on morphotyping of soil-borne spores or on molecular markers, which can be directly applied using colonized roots of the host plant. Until recently, studies of AMF diversity on the population level were impossible, as no suitable marker genes were available. The first population studies on AMF had to rely on DNA from spores or root organ cultures (ROCs) and the molecular markers used could not be applied for the detection of AMF genotypes directly in colonized plant roots from the field. Previous work from our laboratory had shown that mitochondrial ribosomal RNA large subunit gene (mtLSU) sequences are homogeneous within several isolates of Glomus species and that the mitochondrial gene region is a promising marker for distinguishing strains of G. intraradices. The phylotype GLOM A-1 of this morphospecies which was defined in previous studies of our laboratory based on nuclear-encoded rDNA internal transcribed spacers (ITS) sequences seems to occur ubiquitously, showing a high ecological versatility. It is frequently used as model organism and its genome is being sequenced. The aim of this thesis was to develop and apply detection methods based on the mtLSU in order to investigate the diversity of G. intraradices isolates and field populations. The main question was whether this marker is suitable to resolve the genetic structure of this morphospecies which might allow shedding light on the ecological role of strains within the species. In the first part of this thesis, the diversity of the mtLSU was investigated in a set of 16 G. intraradices isolates originating from five continents, either obtained as soil inoculum or as ROC. Among these isolates, 12 different mtLSU haplotypes could be distinguished, whereas homogeneity of the marker within the isolates was confirmed. Several mtLSU haplotypes were already distinguishable by size differences of the PCR products, mainly based on the presence or absence of length-variable introns. The reliability of the marker is dependent on evolutionary intron stability, which was confirmed for some introns by comparisons of multiple culture lineages of the same isolate obtained from different culture collections. In phylogenetic analyses of mtLSU exon sequences from isolates and root-colonizing G. intraradices, several clades could be distinguished. Comparison with ITS sequences from the isolates showed a higher resolution of mtLSU exon sequences which was increased by intron sequences. In order to increase the specificity for G. intraradices and to optimize amplification of the mtLSU fragment from colonized plant roots, a new nested PCR approach was developed and tested using field root samples from a semi-natural grassland and a mine spoil in Hungary. A RFLP approach was developed to reduce time-consuming and expensive cloning and sequencing procedures. In the second part of this thesis, the population structure of an AMF in roots from the environment was analyzed for the first time. Two agricultural field experiments in Switzerland, including different tillage treatments, and two semi-natural grasslands in Switzerland and France were chosen for the investigation of the genetic structure of G. intraradices phylotype GLOM A-1 using the PCR-RFLP approach. Each field site was dominated by one or two frequently found RFLP patterns of G. intraradices GLOM A-1, which were defined as Intra types. The composition of Intra types differed strongly between the agricultural sites and the semi-natural grasslands, but also between the two agricultural sites. In contrast to the situation often found in AMF species community studies, RFLP type richness was higher in the agricultural sites compared to the grasslands. Four Intra types, shared by different sites, were further resolved by sequence analyses, but only the two grasslands were found to share mtLSU sequence haplotypes. In phylogenetic analyses of completely sequenced examples of each Intra type, almost all haplotypes from the grassland sites fell within a separate “grassland clade”. If a single mtLSU haplotype could be specifically detected in a pool of others, such a molecular tool could be used for tracing single strains inoculated in a field site. Nested PCR primers were developed specifically for one single mtLSU haplotype, which dominated one of the agricultural sites and was known from previous studies analyzing ROCs. By applying this approach to all samples from the four study sites, it could be shown that the respective haplotype was only detected in samples previously tested positive for this type using the general approach. In other words, both methods confirmed each other. Two further specific nested PCR approaches were developed for two mtLSU haplotypes representing the G. intraradices isolate BEG140. These approaches were designed to be applied for tracing this isolate inoculated in a field experiment performed in a mine spoil bank of the Czech Republic in the context of a reclamation project. Besides the considerable genetic structure of this fungus among the isolates studied and in the roots of the field sites, evidence of specialized mtLSU haplotypes was reported, which might represent ecotypes or even different (“cryptic”) species. It could be shown that world-wide mtLSU haplotype diversity of G. intraradices is considerably higher than previously assumed. More investigations of different ecosystems are required for the determination of adapted ecotypes. The approaches developed here will be furthermore useful for instance in inoculation experiments and functional tests, e.g. in greenhouse experiments. By presenting first insights into the genetic structure of the most widespread species of arbuscular mycorrhizal fungi, the findings presented here will have major implications on our views of processes of adaptation and specialization in these plant ⁄ fungus associations

What’s bothering developers in code review?

The practice of code review is widely adopted in industry and hasbeen studied to an increasing degree in the research community.However, the developer experience of code review has receivedlimited attention. Here, we report on initial results from a mixed-method exploratory study of the developer experience

Преподавание гигиены: состояние, проблемы, перспективы

ВУЗЫВЫСШИЕ УЧЕБНЫЕ ЗАВЕДЕНИЯОБРАЗОВАНИЕ МЕДИЦИНСКОЕГИГИЕНА (ДИСЦИПЛИНА)ГИГИЕНА /ОБУЧМЕТОДИКА ПРЕПОДАВАНИ

Co-circulation of a novel phlebovirus and Massilia virus in sandflies, Portugal

Free PMC Article: www.ncbi.nlm.nih.gov/pmc/articles/pmid/26497645/Background: In Portugal, entomological surveys to detect phleboviruses in their natural vectors have not been performed so far. Thus, the aims of the present study were to detect, isolate and characterize phleboviruses in sandfly populations of Portugal. Findings: From May to October 2007–2008, 896 female sandflies were trapped in Arrábida region, located on the southwest coast of Portugal. Phlebovirus RNA was detected by using a pan-phlebovirus RT-PCR in 4 out of 34 Phlebotomus perniciosus pools. Direct sequencing of the amplicons showed that 2 samples exhibited 72 % nucleotide identity with Arbia virus, and two showed 96 % nucleotide identity with Massilia virus. The Arbia-like virus (named Alcube virus) was isolated in cell culture and complete genomic sequences of one Alcube and two Massila viruses were determined using next-generation sequencing technology. Phylogenetic analysis demonstrated that Alcube virus clustered with members of the Salehabad virus species complex. Within this clade, Alcube virus forms a monophyletic lineage with the Arbia, Salehabad and Adana viruses sharing a common ancestor. Arbia virus has been identified as the most closely related virus with 20-28 % nucleotide and 10-27 % amino acid divergences depending on the analysed segment. Conclusions: We have provided genetic evidence for the circulation of a novel phlebovirus species named Alcube virus in Ph. perniciosus and co-circulation of Massilia virus, in Arrábida region, southwest of Portugal. Further epidemiological investigations and surveillance for sandfly-borne phleboviruses in Portugal are needed to elucidate their medical importance.This work was partially funded by the FCT project “New arboviruses isolated in Portugal. Risk assessment and public health application" (PTDC/SAU-SAP/119199/2010)

Genetic diversity of the arbuscular mycorrhizal fungus Glomus intraradices as determined by mitochondrial large subunit rRNA gene sequences is considerably higher than previously expected

Summary: • Glomus intraradices is a widespread arbuscular mycorrhizal fungus (AMF), which has been found in an extremely broad range of habitats, indicating a high tolerance for environmental factors and a generalist life history strategy. Despite this ecological versatility, not much is known about the genetic diversity of this fungal species across different habitats or over large geographic scales. • A nested polymerase chain reaction (PCR) approach for the mitochondrial rRNA large subunit gene (mtLSU), distinguished different haplotypes among cultivated isolates of G. intraradices and within mycorrhizal root samples from the field. • From analysis of 16 isolates of this species originating from five continents, 12 mitochondrial haplotypes were distinguished. Five additional mtLSU haplotypes were detected in field‐collected mycorrhizal roots. Some introns in the mtLSU region appear to be stable over years of cultivation and are ancestral to the G. intraradices clade. • Genetic diversity within G. intraradices is substantially higher than previously thought, although some mtLSU haplotypes are widespread. A restriction fragment length polymorphism approach also was developed to distinguish mtLSU haplotypes without sequencing. Using this molecular tool, intraspecific genetic variation of an AMF species can be studied directly in field plants

Population genomics reveals that within-fungus polymorphism is common and maintained in populations of the mycorrhizal fungus Rhizophagus irregularis.

Arbuscular mycorrhizal (AM) fungi are symbionts of most plants, increasing plant growth and diversity. The model AM fungus Rhizophagus irregularis (isolate DAOM 197198) exhibits low within-fungus polymorphism. In contrast, another study reported high within-fungus variability. Experiments with other R. irregularis isolates suggest that within-fungus genetic variation can affect the fungal phenotype and plant growth, highlighting the biological importance of such variation. We investigated whether there is evidence of differing levels of within-fungus polymorphism in an R. irregularis population. We genotyped 20 isolates using restriction site-associated DNA sequencing and developed novel approaches for characterizing polymorphism among haploid nuclei. All isolates exhibited higher within-isolate poly-allelic single-nucleotide polymorphism (SNP) densities than DAOM 197198 in repeated and non-repeated sites mapped to the reference genome. Poly-allelic SNPs were independently confirmed. Allele frequencies within isolates deviated from diploids or tetraploids, or that expected for a strict dikaryote. Phylogeny based on poly-allelic sites was robust and mirrored the standard phylogeny. This indicates that within-fungus genetic variation is maintained in AM fungal populations. Our results predict a heterokaryotic state in the population, considerable differences in copy number variation among isolates and divergence among the copies, or aneuploidy in some isolates. The variation may be a combination of all of these hypotheses. Within-isolate genetic variation in R. irregularis leads to large differences in plant growth. Therefore, characterizing genomic variation within AM fungal populations is of major ecological importance

Potential vectors of equine arboviruses in the UK

There is growing concern about the increasing risk of disease outbreaks caused by arthropod-borne viruses (arboviruses) in both humans and animals. There are several mosquito-borne viral diseases that cause varying levels of morbidity and mortality in horses and that can have substantial welfare and economic ramifications. While none has been recorded in the UK, vector species for some of these viruses are present, suggesting that UK equines may be at risk. We undertook, therefore, the first study of mosquito species on equine premises in the UK. Mosquito Magnet traps and red-box traps were used to sample adults, and larvae were collected from water sources such as tyres, buckets, ditches and pools. Several species which are known to be capable of transmitting important equine infectious arboviruses were trapped. The most abundant, with a maximum catch of 173 in 72 hours was Ochlerotatus detritus, a competent vector of some flaviviruses; the highest densities were found near saltmarsh habitats. The most widespread species, recorded at >75% of sites, was Culiseta annulata. This study demonstrates that potential mosquito vectors of arboviruses, including those known to be capable of infecting horses, are present and may be abundant on equine premises in the UK

An effective method for the identification and separation of Anopheles minimus, the primary malaria vector in Thailand, and its sister species Anopheles harrisoni, with a comparison of their mating behaviors

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The role of community and population ecology in applying mycorrhizal fungi for improved food security.

The global human population is expected to reach ∼9 billion by 2050. Feeding this many people represents a major challenge requiring global crop yield increases of up to 100%. Microbial symbionts of plants such as arbuscular mycorrhizal fungi (AMF) represent a huge, but unrealized resource for improving yields of globally important crops, especially in the tropics. We argue that the application of AMF in agriculture is too simplistic and ignores basic ecological principals. To achieve this challenge, a community and population ecology approach can contribute greatly. First, ecologists could significantly improve our understanding of the determinants of the survival of introduced AMF, the role of adaptability and intraspecific diversity of AMF and whether inoculation has a direct or indirect effect on plant production. Second, we call for extensive metagenomics as well as population genomics studies that are crucial to assess the environmental impact that introduction of non-local AMF may have on native AMF communities and populations. Finally, we plead for an ecologically sound use of AMF in efforts to increase food security at a global scale in a sustainable manner