Resistance and mitotic instability to chloroneb and 1,4-oxathiin in Aspergillus nidulans.

Mutants resistant to two fungicides, chloroneb (1,4-dichloro-2,5-dimethoxybenzene) and vitavax (2,3-dihydro-5-carboxanilido-6-methyl-1,4-oxathiin) were spontaneously obtained from a strain of Aspergillus nidulans with frequencies of 12.5 and 1.1 respectively, in 10(8) conidia. One chloroneb-resistant mutant (Chl 1) segregated as a single gene and was mapped in linkage group IV. It also caused a partial dependence of the strain on the fungicide and was semi-dominant. The mutant resistant to vitavax (Vit 1) also segregated as a single gene and was dominant. Both fungicides altered the instability of diploid and duplication strains. Chloroneb mainly increased haploidization, and vitavax reduced the mitotic recombination in diploids. Chloroneb increased the instability of duplication strains, and vitavax reduced such instability. The possible mode of action of such fungicides affecting stability is discussed.     

Placentation in dolphins from the Amazon River Basin: the Boto, Inia geoffrensis, and the Tucuxi, Sotalia fluviatilis

A recent reassessment of the phylogenetic affinities of cetaceans makes it timely to compare their placentation with that of the artiodactyls. We studied the placentae of two sympatric species of dolphin from the Amazon River Basin, representing two distinct families. The umbilical cord branched to supply a bilobed allantoic sac. Small blood vessels and smooth muscle bundles were found within the stroma of the cord. Foci of squamous metaplasia occurred in the allanto-amnion and allantochorion. The interhemal membrane of the placenta was of the epitheliochorial type. Two different types of trophoblastic epithelium were seen. Most was of the simple columnar type and indented by fetal capillaries. However, there were also areolar regions with tall columnar trophoblast and these were more sparsely supplied with capillaries. The endometrium was well vascularised and richly supplied with actively secreting glands. These findings are consistent with the current view that Cetacea are nested within Artiodactyla as sister group to the hippopotamids.     

Field release of genetically marked Azospirillum brasilense in association with Sorghum bicolor L.

The agronomic impact of genetically tagged azospirilla (Azospirillum brasilense)was assessed in open field and their fluctuation were monitored in the soil/rhizosphere. Strain performance, upon inoculation of sorghum, was evaluated over a two-years period; agronomic treatments included nitrogen application (0, 80, 160 kg ha^–1), and types of inoculant (Sp245 lacZ, Sp6 gusA, Sp6 IAA⁺⁺ gusA). Grain yield was higher for inoculated seed plots than in non-inoculated ones, whereas nitrogen content, biomass of plant residues and nitrogen in plant residues gave values that were not statistically different. Root length density (RLD) of sorghum at the end of the stem elongation stage was affected only by the indole-3-acetic acid (IAA) overproducer Azospirillum strain (A. brasilense Sp6 IAA⁺⁺ gusA) with respect to the normal IAA producer (A. brasilense Sp6 gusA), being higher in the first 40 cm of depth, notwithstanding the level of nitrogen fertilization. The traceability of the released genetically modified strains enabled to monitor their ability to colonise soil and roots. Moreover, the genetic modification per se vs. the non-modified counterpart, did not affect the culturable aerobic population in soil, microfungi, streptomycetes, fluorescent pseudomonads, soil microbial biomass, or some microbial activities, all selected as important indicators.     

Interglacial microrefugia and diversification of a cactus species complex: phylogeography and palaeodistributional reconstructions for Pilosocereus aurisetus and allies

The role of Pleistocene climate changes in promoting evolutionary diversification in global biota is well documented, but the great majority of data regarding this subject come from North America and Europe, which were greatly affected by glaciation. The effects of Pleistocene changes on cold‐ and/or dry‐adapted species in tropical areas where glaciers were not present remain sparsely investigated. Many such species are restricted to small areas surrounded by unfavourable habitats, which may represent potential interglacial microrefugia. Here, we analysed the phylogeographic structure and diversification history of seven cactus species in the Pilosocereus aurisetus complex that are restricted to rocky areas with high diversity and endemism within the Neotropical savannas of eastern South America. We combined palaeodistributional estimates with standard phylogeographic approaches based on two chloroplast DNA regions (trnT‐trnL and trnS‐trnG), exon 1 of the nuclear gene PhyC and 10 nuclear microsatellite loci. Our analyses revealed a phylogeographic history marked by multiple levels of distributional fragmentation, isolation leading to allopatric differentiation and secondary contact among divergent lineages within the complex. Diversification and demographic events appear to have been affected by the Quaternary climatic cycles as a result of isolation in multiple patches of xerophytic vegetation. These small patches presently harbouring P. aurisetus populations seem to operate as microrefugia, both at present and during Pleistocene interglacial periods; the role of such microrefugia should be explored and analysed in greater detail.     

Homologous Hevea brasiliensis REF (Hevb1) and SRPP (Hevb3) present different auto-assembling

HbREF and HbSRPP are two Hevea brasiliensis proteins present on rubber particles, and probably involved in the coagulation of latex. Their function is unclear, but we previously discovered that REF had amyloid properties, which could be of particular interest during the coagulation process. First, we confirmed that REF and SRPP, homologous and principal proteins in hevea latex, are not glycoproteins. In this work, we investigated various aspects of protein interactions: aggregation, auto-assembling, yeast and erythrocyte agglutination, co-interactions by various biochemical (PAGE, spectroscopy, microscopy), biophysical (DLS, ellipsometry) and structural (TEM, ATR-FTIR, PM-IRRAS) approaches. We demonstrated that both proteins are auto-assembling into different aggregative states: REF polymerizes as an amyloid rich in β-sheets and forms quickly large aggregates (> μm), whereas SRPP auto-assembles in solution into stable nanomultimers of a more globular nature. Both proteins are however able to interact together, and SRPP may inhibit the amyloidogenesis of REF. REF is also able to interact with the membranes of yeasts and erythrocytes, leading to their agglutination. In addition, we also showed that both REF and SRPP did not have antimicrobial activity, whereas their activity on membranes has been clearly evidenced. We may suspect that these aggregative properties, even though they are clearly different, may occur during coagulation, when the membrane is destabilized. The interaction of proteins with membranes could help in the colloidal stability of latex, whereas the protein–protein interactions would contribute to the coagulation process, by bringing rubber particles together or eventually disrupting the particle monomembranes.     

Comparative and predictive phylogeography in the South American diagonal of open formations: Unravelling the biological and environmental influences on multitaxon demography

Phylogeography investigates historical drivers of the geographical distribution of intraspecific lineages. Particular attention has been given to ecological, climatic and geological processes in the diversification of the Neotropical biota. Several species sampled across the South American diagonal of open formations (DOF), comprising the Caatinga, Cerrado and Chaco biomes, experienced range shifts coincident with Quaternary climatic changes. However, comparative studies across different spatial, temporal and biological scales on DOF species are still meagre. Here, we combine phylogeographical model selection and machine learning predictive frameworks to investigate the influence of Pleistocene climatic changes on several plant and animal species from the DOF. We assembled mitochondrial/chloroplastic DNA sequences in public repositories and inferred the demographic responses of 44 species, comprising 70 intraspecific lineages of plants, lizards, frogs, spiders and insects. We then built a random forest model using biotic and abiotic information to identify the best predictors of demographic responses in the Pleistocene. Finally, we assessed the temporal synchrony of species demographic responses with hierarchical approximate Bayesian computation. Biotic variables related to population connectivity, gene flow and habitat preferences largely predicted how species responded to Pleistocene climatic changes, and demographic changes were synchronous primarily during the Middle Pleistocene. Although 22 (~31%) lineages underwent demographic expansion, presumably associated with the spread of aridity during the glacial Pleistocene periods, our findings suggest that nine lineages (~13%) exhibited the opposite response due to taxon-specific attributes.     

Isopentenyl diphosphate isomerase: A checkpoint to isoprenoid biosynthesis

Even if the isopentenyl diphosphate (IPP) isomerases have been discovered in the 50s, it is only in the last decade that the genetical, enzymatical, structural richness and cellular importance of this large family of crucial enzymes has been uncovered. Present in all living kingdoms, they can be classified in two subfamilies: type 1 and type 2 IPP isomerases, which show clearly distinct characteristics. They all perform the regulatory isomerization of isopentenyl diphosphate into dimethylallyl diphosphate, a key rate-limiting step of the terpenoid biosynthesis, via a protonation/deprotonation mechanism. Due to their importance in the isoprenoid metabolism and the increasing interest of industry devoted to terpenoid production, it is foreseen that the biotechnological development of such enzymes should be under intense scrutiny in the near future.     

An integrated approach for the evaluation of biological control of the complex Polymyxa betae/Beet Necrotic Yellow Vein Virus,by means of seed inoculants

Rhizomania is an extremely severe sugarbeet disease caused by the complex Polymyxa betae/Beet Necrotic Yellow Vein Virus (BNYVV). A relatively small number of recently introduced sugarbeet cultivars characterized by a high tolerance to rhizomania are available on the market. An integrated approach was therefore developed using Pseudomonas fluorescens biological control agents (BCAs) in order to improve yield performance of cultivars characterized by a medium tolerance to the disease. A genetically modified biological control agent, Pseudomonas fluorescens F113Rif (pCUGP), was developed for enhanced production of the antimicrobial metabolite 2,4-diacetylphloroglucinol (Phl) and lacking an antibiotic resistance marker gene, making the strain suitable for field release. The ability of synthetic Phl and P. fluorescens F113Rif (pCUGP) to antagonize the fungal vector, P. betae, was assessed in microcosm trials. Results encouraged the preparation of multiple field trials in a soil naturally infested with P. betae/BNYVV, to determine the biocontrol efficacy of P. fluorescens F113Rif (pCUGP) and to assess its impact on sugarbeet yield and quality and on the indigenous microbial population. While the colonization ability of P. fluorescens F113Rif (pCUGP) was satisfactory at sugarbeet emergence (2.5×10⁶ CFU g^–1 root), control of rhizomania was not achieved. Inoculation of sugarbeet with Pseudomonas fluorescens F113Rif (pCUGP) did not affect crop yield and quality nor affect the numbers of selected microbial populations.     

Using Next Generation RAD Sequencing to Isolate Multispecies Microsatellites for Pilosocereus (Cactaceae)

Microsatellite markers (also known as SSRs, Simple Sequence Repeats) are widely used in plant science and are among the most informative molecular markers for population genetic investigations, but the development of such markers presents substantial challenges. In this report, we discuss how next generation sequencing can replace the cloning, Sanger sequencing, identification of polymorphic loci, and testing cross-amplification that were previously required to develop microsatellites. We report the development of a large set of microsatellite markers for five species of the Neotropical cactus genus Pilosocereus using a restriction-site-associated DNA sequencing (RAD-seq) on a Roche 454 platform. We identified an average of 165 microsatellites per individual, with the absolute numbers across individuals proportional to the sequence reads obtained per individual. Frequency distribution of the repeat units was similar in the five species, with shorter motifs such as di- and trinucleotide being the most abundant repeats. In addition, we provide 72 microsatellites that could be potentially amplified in the sampled species and 22 polymorphic microsatellites validated in two populations of the species Pilosocereus machrisii. Although low coverage sequencing among individuals was observed for most of the loci, which we suggest to be more related to the nature of the microsatellite markers and the possible bias inserted by the restriction enzymes than to the genome size, our work demonstrates that an NGS approach is an efficient method to isolate multispecies microsatellites even in non-model organisms.