Diversity of Leaf Fungal Endophytes from Two Coffea arabica Varieties and Antagonism towards Coffee Leaf Rust.

Coffee has immense value as a worldwide-appreciated commodity. However, its production faces the effects of climate change and the spread of severe diseases such as coffee leaf rust (CLR). The exploration of fungal endophytes associated with Coffea sp. has already found the existence of nearly 600 fungal species, but their role in the plants remains practically unknown. We have researched the diversity of leaf fungal endophytes in two Coffea arabica varieties: one susceptible and one resistant to CLR. Then, we conducted cross-infection essays with four common endophyte species (three Colletotrichum sp. and Xylaria sp. 1) and Hemileia vastatrix (CLR) in leaf discs, to investigate the interaction of the endophytes on CLR colonisation success and severity of infection. Two Colletotrichum sp., when inoculated 72 h before H. vastatrix, prevented the colonisation of the leaf disc by the latter. Moreover, the presence of endophytes prior to the arrival of H. vastatrix ameliorated the severity of CLR. Our work highlights both the importance of characterising the hidden biodiversity of endophytes and investigating their potential roles in the plant-endophyte interaction.

Heterogeneous effects of climatic conditions on Andean bean landraces and cowpeas highlight alternatives for crop management and conservation.

The use and conservation of agrobiodiversity have become critical to face the actual and future challenges imposed by climate change. Collecting phytogenetic resources is a first step for their conservation; however, the genetic material must be analysed to understand their potential to improve agricultural resilience and adaptation to the new climatic conditions. We have selected nine Phaseolus vulgaris, one P. lunatus and two Vigna unguiculata landraces from two different climatic backgrounds of the Andean region of South Ecuador and one P. vulgaris commercial cultivar, and we grew them under two different conditions of temperature and humidity (open field and greenhouse). Then, we recorded data for 32 characters of plant architecture, flower and fruit characteristics and yield, and 17 events in the phenology of the plants. We analysed the impact of treatment on species, climatic background, and each of the landraces, and identified both characters and landraces that are mostly affected by changes in their environmental conditions. Overall, higher temperatures were benign for all materials except for two P. vulgaris landraces from cold background, which performed better or developed faster under cold conditions. Finally, we calculated a climate resilience landrace index, which allowed us to classify the landraces by their plasticity to new environmental conditions, and found heterogeneous landrace susceptibility to warmer conditions. Two P. vulgaris landraces were highlighted as critical targets for conservation.

Do Host Plant and Associated Ant Species Affect Microbial Communities in Myrmecophytes?

Ant-associated microorganisms can play crucial and often overlooked roles, and given the diversity of interactions that ants have developed, the study of the associated microbiomes is of interest. We focused here on specialist plant-ant species of the genus Allomerus that grow a fungus to build galleries on their host-plant stems. Allomerus-inhabited domatia, thus, might be a rich arena for microbes associated with the ants, the plant, and the fungus. We investigated the microbial communities present in domatia colonised by four arboreal ants: Allomerus decemarticulatus, A. octoarticulatus, A. octoarticulatus var. demerarae, and the non-fungus growing plant-ant Azteca sp. cf. depilis, inhabiting Hirtella physophora or Cordia nodosa in French Guiana. We hypothesized that the microbial community will differ among these species. We isolated microorganisms from five colonies of each species, sequenced the 16S rRNA or Internal TranscribedSpacer (ITS) regions, and described both the alpha and beta diversities. We identified 69 microbial taxa, which belong to five bacterial and two fungal phyla. The most diverse phyla were Proteobacteria and Actinobacteria. The microbial community of Azteca cf. depilis and Allomerus spp. differed in composition and richness. Geographical distance affected microbial communities and richness but plant species did not. Actinobacteria were only associated with Allomerus spp.

Spanish list of potentially inappropriate drugs in the elderly (ES-PIA project).

PURPOSE: In the last decades, different criteria have been developed for detecting inappropriate prescription in older patients. In Spain, translations and adaptations of international lists are available but it would be necessary a national list which could cope with the peculiarities of our health system, existing pharmaceutical market, and prescription habits. We propose in this project the creation of a Spanish potentially inappropriate drugs list which could be applicable in our clinical scenario. METHODS: We use a Delphi method involving 25 experts from different backgrounds (Clinical Pharmacology, Geriatrics, Rational Use of Drugs and Pharmacy, Primary Care and Pharmacoepidemiology, and Pharmacovigilance) that were asked to participate in two-round questionnaires. For analysis, current recommendations of Worth and Pigni were applied, and every statement was classified into one of three groups: strong, moderate, or low agreement. Statements with strong agreement were accepted to be part of the inadequate prescription list. Moderate agreement statements were selected to enter the second questionnaire, and statements with low agreement were further analyzed to determine if it was due to heterogeneity or due to dispersion in the answers. RESULTS: The first questionnaire consisted of 160 proposed sentences, of which 106 reached a high agreement, 32 a moderate agreement, and 22 a low agreement. All sentences proposed in the second questionnaire reached a strong agreement. The total accepted sentences were 138. CONCLUSIONS: We offer a list of inadequate prescription in older patients adapted to the Spanish pharmacopeia and according to the prescription habits in our environment.

Arabidopsis Heat Stress-Induced Proteins Are Enriched in Electrostatically Charged Amino Acids and Intrinsically Disordered Regions.

Comparison of the proteins of thermophilic, mesophilic, and psychrophilic prokaryotes has revealed several features characteristic to proteins adapted to high temperatures, which increase their thermostability. These characteristics include a profusion of disulfide bonds, salt bridges, hydrogen bonds, and hydrophobic interactions, and a depletion in intrinsically disordered regions. It is unclear, however, whether such differences can also be observed in eukaryotic proteins or when comparing proteins that are adapted to temperatures that are more subtly different. When an organism is exposed to high temperatures, a subset of its proteins is overexpressed (heat-induced proteins), whereas others are either repressed (heat-repressed proteins) or remain unaffected. Here, we determine the expression levels of all genes in the eukaryotic model system Arabidopsis thaliana at 22 and 37 °C, and compare both the amino acid compositions and levels of intrinsic disorder of heat-induced and heat-repressed proteins. We show that, compared to heat-repressed proteins, heat-induced proteins are enriched in electrostatically charged amino acids and depleted in polar amino acids, mirroring thermophile proteins. However, in contrast with thermophile proteins, heat-induced proteins are enriched in intrinsically disordered regions, and depleted in hydrophobic amino acids. Our results indicate that temperature adaptation at the level of amino acid composition and intrinsic disorder can be observed not only in proteins of thermophilic organisms, but also in eukaryotic heat-induced proteins; the underlying adaptation pathways, however, are similar but not the same.

The impact of an extreme climatic disturbance and different fertilization treatments on plant development, phenology, and yield of two cultivar groups of Solanum betaceum Cav.

Changing climatic conditions impose a challenge both to biodiversity and food security. The effects of climate change affect different aspects of the plant or crop, such as morphological and phenological aspects, as well as yield. The effects of greenhouse conditions might be comparable in some cases to a permanent extreme disturbance in climate and weather, thus, contributing to our knowledge on climate change impacts on plant species. We have investigated the differences for 23 traits in two cultivar groups of an Andean traditional crop, Solanum betaceum, under two different environmental conditions that correspond to the traditional practices in the open field and three cultural managements under greenhouse conditions (no fertilization or control, organic, and mineral). We found that traditional practices in the open field are the less productive. Moreover, in warmer and drier conditions the treatment with organic fertilization was the most productive. Greenhouse conditions, however, delay production. We further identified traits that differentiate both cultivar groups and traits that are linked to either the new climate conditions or the fertilization treatments. Fruit characteristics were quite homogeneous between the two cultivar groups. Overall, our results provide insight on the consequences that climate change effects might exert on crops such as tree tomato, reveal that greenhouses can be a robust alternative for tree tomato production, and highlight the need to understand how different managements are linked to different solutions to fulfil the farmers' demands.

Essentiality Is a Strong Determinant of Protein Rates of Evolution during Mutation Accumulation Experiments in Escherichia coli.

The Neutral Theory of Molecular Evolution is considered the most powerful theory to understand the evolutionary behavior of proteins. One of the main predictions of this theory is that essential proteins should evolve slower than dispensable ones owing to increased selective constraints. Comparison of genomes of different species, however, has revealed only small differences between the rates of evolution of essential and nonessential proteins. In some analyses, these differences vanish once confounding factors are controlled for, whereas in other cases essentiality seems to have an independent, albeit small, effect. It has been argued that comparing relatively distant genomes may entail a number of limitations. For instance, many of the genes that are dispensable in controlled lab conditions may be essential in some of the conditions faced in nature. Moreover, essentiality can change during evolution, and rates of protein evolution are simultaneously shaped by a variety of factors, whose individual effects are difficult to isolate. Here, we conducted two parallel mutation accumulation experiments in Escherichia coli, during 5,500-5,750 generations, and compared the genomes at different points of the experiments. Our approach (a short-term experiment, under highly controlled conditions) enabled us to overcome many of the limitations of previous studies. We observed that essential proteins evolved substantially slower than nonessential ones during our experiments. Strikingly, rates of protein evolution were only moderately affected by expression level and protein length.

Resistance of Feather-Associated Bacteria to Intermediate Levels of Ionizing Radiation near Chernobyl.

Ionizing radiation has been shown to produce negative effects on organisms, although little is known about its ecological and evolutionary effects. As a study model, we isolated bacteria associated with feathers from barn swallows Hirundo rustica from three study areas around Chernobyl differing in background ionizing radiation levels and one control study site in Denmark. Each bacterial community was exposed to four different γ radiation doses ranging from 0.46 to 3.96 kGy to test whether chronic exposure to radiation had selected for resistant bacterial strains. Experimental radiation duration had an increasingly overall negative effect on the survival of all bacterial communities. After exposure to γ radiation, bacteria isolated from the site with intermediate background radiation levels survived better and produced more colonies than the bacterial communities from other study sites with higher or lower background radiation levels. Long-term effects of radiation in natural populations might be an important selective pressure on traits of bacteria that facilitate survival in certain environments. Our findings indicate the importance of further studies to understand the proximate mechanisms acting to buffer the negative effects of ionizing radiation in natural populations.

Coevolution analyses illuminate the dependencies between amino acid sites in the chaperonin system GroES-L.

BACKGROUND: GroESL is a heat-shock protein ubiquitous in bacteria and eukaryotic organelles. This evolutionarily conserved protein is involved in the folding of a wide variety of other proteins in the cytosol, being essential to the cell. The folding activity proceeds through strong conformational changes mediated by the co-chaperonin GroES and ATP. Functions alternative to folding have been previously described for GroEL in different bacterial groups, supporting enormous functional and structural plasticity for this molecule and the existence of a hidden combinatorial code in the protein sequence enabling such functions. Describing this plasticity can shed light on the functional diversity of GroEL. We hypothesize that different overlapping sets of amino acids coevolve within GroEL, GroES and between both these proteins. Shifts in these coevolutionary relationships may inevitably lead to evolution of alternative functions. RESULTS: We conducted the first coevolution analyses in an extensive bacterial phylogeny, revealing complex networks of evolutionary dependencies between residues in GroESL. These networks differed among bacterial groups and involved amino acid sites with functional importance and others with previously unsuspected functional potential. Coevolutionary networks formed statistically independent units among bacterial groups and map to structurally continuous regions in the protein, suggesting their functional link. Sites involved in coevolution fell within narrow structural regions, supporting dynamic combinatorial functional links involving similar protein domains. Moreover, coevolving sites within a bacterial group mapped to regions previously identified as involved in folding-unrelated functions, and thus, coevolution may mediate alternative functions. CONCLUSIONS: Our results highlight the evolutionary plasticity of GroEL across the entire bacterial phylogeny. Evidence on the functional importance of coevolving sites illuminates the as yet unappreciated functional diversity of proteins.

An efficient protocol for isolating melanised chaetothyrialean anamorphic fungi associated with plant-ants.

Because of their ecological characteristics, slow growth rates and the presence of contaminants, Chaetothyriales fungi associated with structures built by tropical plant-ants can be difficult to isolate with standard procedures. Here, we describe an easy-to-use protocol for obtaining pure cultures by using cotton as a first substrate. We have further found by means of fluorescent stains that nuclei concentrate either in young hyphae or in the tips of the hyphae.

Dynamic transmission, host quality, and population structure in a multihost parasite of bumblebees.

The evolutionary ecology of multihost parasites is predicted to depend upon patterns of host quality and the dynamics of transmission networks. Depending upon the differences in host quality and transmission asymmetries, as well as the balance between intra- and interspecific transmission, the evolution of specialist or generalist strategies is predicted. Using a trypanosome parasite of bumblebees, we ask how host quality and transmission networks relate to parasite population structure across host species, and thus the potential for the evolution of specialist strains adapted to different host species. Host species differed in quality, with parasite growth varying across host species. Highly asymmetric transmission networks, together with differences in host quality, likely explain local population structure of the parasite across host species. However, parasite population structure across years was highly dynamic, with parasite populations varying significantly from one year to the next within individual species at a given site. This suggests that, while host quality and transmission may provide the opportunity for short-term host specialization by the parasite, repeated bottlenecking of the parasite, in combination with its own reproductive biology, overrides these smaller scale effects, resulting in the evolution of a generalist parasite.

Fungus-growing Allomerus ants are associated with antibiotic-producing actinobacteria.

Fungus-growing attine ants use natural-product antibiotics produced by mutualist actinobacteria as 'weedkillers' in their fungal gardens. Here we report for the first time that fungus-growing Allomerus ants, which lie outside the tribe Attini, are associated with antifungal-producing actinobacteria, which offer them protection against non-cultivar fungi isolated from their ant-plants.

Protein coadaptation and the design of novel approaches to identify protein-protein interactions.

Proteins rarely function in isolation but they form part of complex networks of interactions with other proteins within or among cells. The importance of a particular protein for cell viability is directly dependent upon the number of interactions where it participates and the function it performs: the larger the number of interactions of a protein the greater its functional importance is for the cell. With the advent of genome sequencing and "omics" technologies it became feasible conducting large-scale searches for protein interacting partners. Unfortunately, the accuracy of such analyses has been underwhelming owing to methodological limitations and to the inherent complexity of protein interactions. In addition to these experimental approaches, many computational methods have been developed to identify protein-protein interactions by assuming that interacting proteins coevolve resulting from the coadaptation dynamics between the amino acids of their interacting faces. We review the main technological advances made in the field of interactomics and discuss the feasibility of computational methods to identify protein-protein interactions based on the estimation of coevolution. As proof-of-concept, we present a classical case study: the interactions of cell surface proteins (receptors) and their ligands. Finally, we take this discussion one step forward to include interactions between organisms and species to understand the generation of biological complexity. Development of technologies for accurate detection of protein-protein interactions may shed light on processes that go from the fine-tuning of pathways and metabolic networks to the emergence of biological complexity.

Specific, non-nutritional association between an ascomycete fungus and Allomerus plant-ants.

Ant-fungus associations are well known from attine ants, whose nutrition is based on a symbiosis with basidiomycete fungi. Otherwise, only a few non-nutritional ant-fungus associations have been recorded to date. Here we focus on one of these associations involving Allomerus plant-ants that build galleried structures on their myrmecophytic hosts in order to ambush prey. We show that this association is not opportunistic because the ants select from a monophyletic group of closely related fungal haplotypes of an ascomycete species from the order Chaetothyriales that consistently grows on and has been isolated from the galleries. Both the ants' behaviour and an analysis of the genetic population structure of the ants and the fungus argue for host specificity in this interaction. The ants' behaviour reveals a major investment in manipulating, growing and cleaning the fungus. A molecular analysis of the fungus demonstrates the widespread occurrence of one haplotype and many other haplotypes with a lower occurrence, as well as significant variation in the presence of these fungal haplotypes between areas and ant species. Altogether, these results suggest that such an interaction might represent an as-yet undescribed type of specific association between ants and fungus in which the ants cultivate fungal mycelia to strengthen their hunting galleries.

New findings in insect fungiculture: Have ants developed non-food, agricultural products?

The interaction between Allomerus plant-ants and an ascomycete fungus growing on and strengthening their galleries is not opportunistic. We previously demonstrated that this association is highly specific as only one fungal species represented by a few haplotypes was found associated with the ants. We also discovered that the ants' behavior revealed a major investment in manipulating and enhancing the growth of their associated fungus. We have growing evidence that this specificity is consistent with selection by the ants. Here, we discuss this selection within the framework of insect agriculture, as we believe these ants fulfill all of the prerequisites to be considered as farmers. Allomerus ants promote their symbiont's growth, protect it from potential pathogens and select specific cultivars. Taken together, we think that the interaction between Allomerus ants and their cultivar might represent the first case of insect fungiculture used as a means of obtaining building material.

Rapid induction of immune density-dependent prophylaxis in adult social insects.

The innate immune system provides defence against parasites and pathogens. This defence comes at a cost, suggesting that immune function should exhibit plasticity in response to variation in environmental threats. Density-dependent prophylaxis (DDP) has been demonstrated mostly in phase-polyphenic insects, where larval group size determines levels of immune function in either adults or later larval instars. Social insects exhibit extreme sociality, but DDP has been suggested to be absent from these ecologically dominant taxa. Here we show that adult bumble-bee workers (Bombus terrestris) exhibit rapid plasticity in their immune function in response to social context. These results suggest that DDP does not depend upon larval conditions, and is likely to be a widespread and labile response to rapidly changing conditions in adult insect populations. This has obvious ramifications for experimental analysis of immune function in insects, and serious implications for our understanding of the epidemiology and impact of pathogens and parasites in spatially structured adult insect populations.

Proteasome-related HslU and HslV genes typical of eubacteria are widespread in eukaryotes.

Many eubacteria contain an ATP-dependent protease complex, which is built by multiple copies of the HslV and HslU proteins and is therefore called HslVU. HslU proteins are AAA + ATPases, while HslV proteins are proteases that show highly significant similarity to beta subunits of proteasomes. Therefore, the HslVU complex has been envisaged as a precursor or ancestral type of proteasome. Here we show that species of most of the main eukaryotic lineages have HslU and HslV genes very similar to those found in proteobacteria. We have detected them in amoebozoa, plantae, chromoalveolata, rhizaria, and excavata species. Phylogenetic analyses suggest that these genes have been obtained by endosymbiosis from the proteobacterial ancestor that gave rise to eukaryotic mitochondria. The products encoded by these eukaryotic genes adopt, according to modeling based on the known crystal structures of prokaryotic HslU and HslV proteins, conformations that are compatible with their being fully active, suggesting that functional HslVU complexes may be present in many eukaryotic species.

New insights into the evolutionary history of type 1 rhodopsins.

Type 1 (archaeal) rhodopsins and related rhodopsin-like proteins had been described in a few halophile archaea, gamma-proteobacteria, a single cyanobacteria, some fungi, and a green alga. In exhaustive database searches, we detected rhodopsin-related sequences derived not only from additional fungal species but also from organisms belonging to three groups in which opsins had hitherto not been described: the alpha-proteobacterium Magnetospirillum magnetotacticum, the cryptomonad alga Guillardia theta, and the dinoflagellate Pyrocystis lunula. Putative plant and human type 1 rhodopsin sequences found in the databases are demonstrated to be contaminants of fungal origin. However, a highly diverged sequence supposedly from the plant Oryza sativa was found that is, together with the Pyrocystis sequence, quite similar to gamma-proteobacterial rhodopsins. These close relationships suggest that at least one horizontal gene transfer event involving rhodopsin genes occurred between prokaryotes and eukaryotes. Alternative hypotheses to explain the current phylogenetic range of type 1 rhodopsins are suggested. The broader phylogenetic range found is compatible with an ancient origin of type 1 rhodopsins, their patchy distribution being caused by losses in multiple lineages. However, the possibility of ancient horizontal transfer events between distant relatives cannot be dismissed.