Phenotypic and Genotypic Characterization of Phytophthora infestans Isolates Associated with Tomato and Potato Crops in Colombia.

Late blight disease, caused by the plant pathogen Phytophthora infestans, is one of the major threats for tomato and potato crops. Monitoring the populations of P. infestans is important to determine if there are changes in the sensitivity to fungicides and host preference. In this study, microsatellite markers and mitochondrial haplotypes were used to assess the genotype of isolates of P. infestans collected from tomato and potato plants in Colombia. Furthermore, sensitivity to the three fungicides cymoxanil (penetrant fungicide), mefenoxam, and fluopicolide (systemic fungicides), and tomato-potato host preference, were evaluated. Mitochondrial haplotyping showed that isolates collected on tomato were from the genetic groups Ia and Ib, while isolates collected on potatoes belonged to group IIa. Microsatellite analyses showed that isolates from tomato form two groups, including the Ib mitochondrial haplotype (which is genetically close to the US-1 clonal lineage) and the Ia haplotype (related to the EC-3 lineage), whereas Colombian isolates from potato formed a separate group. Furthermore, differences in sensitivity to fungicides were observed. Eighty-one percent of the isolates tested were resistant to mefenoxam with an EC50 >10 µg ml-1. Forty-two percent of the isolates showed an intermediate resistance to cymoxanil. The EC50 values ranged between 1 and 10 µg ml-1. For fluopicolide, 90% of the isolates were sensitive, with EC50 <1 µg ml-1. Host preference assays showed that potato isolates infected both host species. Thus, isolates that infect potatoes may pose a risk for tomato crops nearby.

Using the Controversy over Human Race to Introduce Students to the Identification and the Evaluation of Arguments.

The identification and the evaluation of arguments are fundamental elements of critical thinking. However, the explicit promotion of these elements is virtually absent from university science courses. Much of the reason for this is that in most universities, across nearly all disciplines, instructors are required to see the conceptual content coverage of the syllabus as a priority. Moreover, lack of preparation and the fact that critical thinking activities are time-consuming rapidly reduce the interest of many instructors to include them in their courses. Here, we describe the use of a dialogue-based critical thinking classroom scenario (CTCS). The study used a mixed-methods approach with both quantitative and qualitative analyses of questionnaire responses. One hundred and seventeen undergraduates (73 females; 44 males; ages 16-24 years), enrolled in an introductory science course in Colombia, were asked to identify and evaluate arguments regarding a dialogue between two scientists who explore the controversial question of whether or not the concept of race is applicable to humans. It was found that the dialogue-based CTCS provided students with opportunities to identify and evaluate arguments both for and against the question and to make informed decisions about whether or not the concept of race in humans is biologically meaningful. Moreover, analyses of responses to closed-ended and open-ended questions revealed that more than half the participants were able to evaluate arguments in a fair-minded way. Practical implications for the cultivation of critical thinking skills in higher education and further research are discussed.

Towards Covid-19 Literacy: Investigating the Literacy Levels of University Students in Colombia.

Covid-19 literacy, induced by the coronavirus disease (2019), is characterized as the understanding of Covid-19 as well as informed decisions based upon this understanding. This type of literacy is closely related to health literacy, scientific literacy, and scientific media literacy. It may be obvious to say that Covid-19 literacy is a key factor for governments to effectively manage the Covid-19 transition. However, lack of literature exists about Covid-19 literacy among university students. Therefore, the current study aimed to determine the Covid-19 literacy level among 4168 students from a Colombian university. The data were derived from students' responses to a 25-item anonymous online self-reporting questionnaire. We found that 21-25-year age group, graduate students, students enrolled prior to 2015, and medical students had a significantly higher mean score. Moreover, the Internet (86.8%) was the most popular source of information from which participants gained most information regarding Covid-19. Furthermore, 58.5% of the participants considered health workers as a source that can provide accurate information. Most importantly, the findings reveal the students' knowledge about (1) the role of an eventual process of vaccination, (2) the test currently used as diagnostic for Covid-19, and (3) the fatality rate, three aspects of Covid-19 literacy that deserve more attention. The findings provide a useful basis for the formulation of policies and concrete actions in improving Covid-19 literacy.

Differential Susceptibility of Tree Tomato (Solanum betaceum) Cultivars to Late Blight Caused by Phytophthora betacei.

Host-pathogen interactions of a new species of Phytophthora, causal agent of late blight of tree tomato (Solanum betaceum Cav.), identified as Phytophthora betacei, were investigated with four different cultivars. Thirty-six P. betacei isolates, collected from southern Colombia between 2008 and 2009, were used to inoculate common tree tomato cultivars, Común, Híbrido, Injerto, and Holandés. Data on incubation and latent periods as well as infection efficiency, lesion development, and total sporulation were collected via detached leaf assays. Significant differences in susceptibility, based on the parameters measured, were observed. Común was the most susceptible cultivar, followed by Injerto, Híbrido, and Holandés. The mean incubation period was lowest for Común at 125.6 h post-inoculation (hpi) and highest for Híbrido at 139.4 hpi. No significant differences in latent period were observed. All 36 isolates produced necrotic lesions on Común, and 33, 24, and 21 caused infection on Injerto, Híbrido, and Holandés, respectively. Two isolates were able to cause infection only on Común, and 13 isolates were able to infect all four cultivars. Infection efficiency was significantly higher for the cultivar Común, followed by Injerto, Híbrido, and Holandés. Average lesion size was larger on Común than on any other cultivar. An inverse relationship of lesion size and total sporulation was observed. Común had significantly lower total sporulation than Híbrido and Holandés, which had the smallest average lesion sizes. These data show variation in pathogenicity of P. betacei isolates, under controlled conditions, and differential susceptibility of four distinct S. betaceum cultivars.

Is the Phenomenon of Mefenoxam-Acquired Resistance in Phytophthora infestans Universal?

Phytophthora infestans is the causal agent of late blight disease of potatoes and tomatoes. This disease causes devastating economic losses each year, and control is mainly achieved by the use of fungicides. Unfortunately, populations of P. infestans resistant to fungicides have been documented. Furthermore, studies have reported that sensitive isolates to the phenylamide fungicide, mefenoxam, become less sensitive in vitro after a single passage through sublethal concentrations of fungicide-amended medium. The first objective of this study was to investigate if isolates of P. infestans are capable of acquiring resistance to two additional systemic fungicides, fluopicolide (benzamide) and cymoxanil (cyanoacetamide-oxime). In contrast to the situation with mefenoxam, exposure of isolates to sublethal concentrations of fluopicolide and cymoxanil did not induce reduced sensitivity to these two fungicides. The second objective was to assess if reduced sensitivity to mefenoxam could occur in naturally sensitive isolates of other Phytophthora species and of Phytopythium sp., another oomycete plant pathogen. All Phytophthora spp. assessed (P. infestans, P. betacei, and P. pseudocryptogea) as well as Phytopythium sp. acquired resistance to mefenoxam after previous exposure through medium containing 1 µg ml-1 of mefenoxam. Interestingly, isolate 66 of Phytopythium sp. and the isolate of P. pseudocryptogea tested do not seem to be acquiring resistance to mefenoxam after exposure to medium containing 5 µg ml-1 of this fungicide. The tested isolates of P. palmivora and P. cinnamomi were extremely sensitive to mefenoxam, and thus it was not possible to perform a second transfer to access acquisition of resistance to this fungicide.

Gene regulatory networks on transfer entropy (GRNTE): a novel approach to reconstruct gene regulatory interactions applied to a case study for the plant pathogen Phytophthora infestans.

BACKGROUND: The increasing amounts of genomics data have helped in the understanding of the molecular dynamics of complex systems such as plant and animal diseases. However, transcriptional regulation, although playing a central role in the decision-making process of cellular systems, is still poorly understood. In this study, we linked expression data with mathematical models to infer gene regulatory networks (GRN). We present a simple yet effective method to estimate transcription factors' GRNs from transcriptional data. METHOD: We defined interactions between pairs of genes (edges in the GRN) as the partial mutual information between these genes that takes into account time and possible lags in time from one gene in relation to another. We call this method Gene Regulatory Networks on Transfer Entropy (GRNTE) and it corresponds to Granger causality for Gaussian variables in an autoregressive model. To evaluate the reconstruction accuracy of our method, we generated several sub-networks from the GRN of the eukaryotic yeast model, Saccharomyces cerevisae. Then, we applied this method using experimental data of the plant pathogen Phytophthora infestans. We evaluated the transcriptional expression levels of 48 transcription factors of P. infestans during its interaction with one moderately resistant and one susceptible cultivar of yellow potato (Solanum tuberosum group Phureja), using RT-qPCR. With these data, we reconstructed the regulatory network of P. infestans during its interaction with these hosts. RESULTS: We first evaluated the performance of our method, based on the transfer entropy (GRNTE), on eukaryotic datasets from the GRNs of the yeast S. cerevisae. Results suggest that GRNTE is comparable with the state-of-the-art methods when the parameters for edge detection are properly tuned. In the case of P. infestans, most of the genes considered in this study, showed a significant change in expression from the onset of the interaction (0 h post inoculum - hpi) to the later time-points post inoculation. Hierarchical clustering of the expression data discriminated two distinct periods during the infection: from 12 to 36 hpi and from 48 to 72 hpi for both the moderately resistant and susceptible cultivars. These distinct periods could be associated with two phases of the life cycle of the pathogen when infecting the host plant: the biotrophic and necrotrophic phases. CONCLUSIONS: Here we presented an algorithmic solution to the problem of network reconstruction in time series data. This analytical perspective makes use of the dynamic nature of time series data as it relates to intrinsically dynamic processes such as transcription regulation, were multiple elements of the cell (e.g., transcription factors) act simultaneously and change over time. We applied the algorithm to study the regulatory network of P. infestans during its interaction with two hosts which differ in their level of resistance to the pathogen. Although the gene expression analysis did not show differences between the two hosts, the results of the GRN analyses evidenced rewiring of the genes' interactions according to the resistance level of the host. This suggests that different regulatory processes are activated in response to different environmental cues. Applications of our methodology showed that it could reliably predict where to place edges in the transcriptional networks and sub-networks. The experimental approach used here can help provide insights on the biological role of these interactions on complex processes such as pathogenicity. The code used is available at https://github.com/jccastrog/GRNTE under GNU general public license 3.0.

A Genome-Scale Metabolic Reconstruction of Phytophthora infestans With the Integration of Transcriptional Data Reveals the Key Metabolic Patterns Involved in the Interaction of Its Host.

Phytophthora infestans, the causal agent of late blight disease, affects potatoes and tomatoes worldwide. This plant pathogen has a hemibiotrophic lifestyle, having an initial biotrophic infection phase during which the pathogen spreads within the host tissue, followed by a necrotrophic phase in which host cell death is induced. Although increasing information is available on the molecular mechanisms, underlying the distinct phases of the hemibiotrophic lifestyle, studies that consider the entire metabolic processes in the pathogen while undergoing the biotrophic, transition to necrotrophic, and necrotrophic phases have not been conducted. In this study, the genome-scale metabolic reconstruction of P. infestans was achieved. Subsequently, transcriptional data (microarrays, RNA-seq) was integrated into the metabolic reconstruction to obtain context-specific (metabolic) models (CSMs) of the infection process, using constraint-based reconstruction and analysis. The goal was to identify specific metabolic markers for distinct stages of the pathogen's life cycle. Results indicate that the overall metabolism show significant changes during infection. The most significant changes in metabolism were observed at the latest time points of infection. Metabolic activity associated with purine, pyrimidine, fatty acid, fructose and mannose, arginine, glycine, serine, and threonine amino acids appeared to be the most important metabolisms of the pathogen during the course of the infection, showing high number of reactions associated with them and expression switches at important stages of the life cycle. This study provides a framework for future throughput studies of the metabolic changes during the hemibiotrophic life cycle of this important plant pathogen.

Network Analyses in Plant Pathogens.

Even in the age of big data in Biology, studying the connections between the biological processes and the molecular mechanisms behind them is a challenging task. Systems biology arose as a transversal discipline between biology, chemistry, computer science, mathematics, and physics to facilitate the elucidation of such connections. A scenario, where the application of systems biology constitutes a very powerful tool, is the study of interactions between hosts and pathogens using network approaches. Interactions between pathogenic bacteria and their hosts, both in agricultural and human health contexts are of great interest to researchers worldwide. Large amounts of data have been generated in the last few years within this area of research. However, studies have been relatively limited to simple interactions. This has left great amounts of data that remain to be utilized. Here, we review the main techniques in network analysis and their complementary experimental assays used to investigate bacterial-plant interactions. Other host-pathogen interactions are presented in those cases where few or no examples of plant pathogens exist. Furthermore, we present key results that have been obtained with these techniques and how these can help in the design of new strategies to control bacterial pathogens. The review comprises metabolic simulation, protein-protein interactions, regulatory control of gene expression, host-pathogen modeling, and genome evolution in bacteria. The aim of this review is to offer scientists working on plant-pathogen interactions basic concepts around network biology, as well as an array of techniques that will be useful for a better and more complete interpretation of their data.

Lipid Metabolic Versatility in Malassezia spp. Yeasts Studied through Metabolic Modeling.

Malassezia species are lipophilic and lipid-dependent yeasts belonging to the human and animal microbiota. Typically, they are isolated from regions rich in sebaceous glands. They have been associated with dermatological diseases such as seborrheic dermatitis, pityriasis versicolor, atopic dermatitis, and folliculitis. The genomes of Malassezia globosa, Malassezia sympodialis, and Malassezia pachydermatis lack the genes related to fatty acid synthesis. Here, the lipid-synthesis pathways of these species, as well as of Malassezia furfur, and of an atypical M. furfur variant were reconstructed using genome data and Constraints Based Reconstruction and Analysis. To this end, the genomes of M. furfur CBS 1878 and the atypical M. furfur 4DS were sequenced and annotated. The resulting Enzyme Commission numbers and predicted reactions were similar to the other Malassezia strains despite the differences in their genome size. Proteomic profiling was utilized to validate flux distributions. Flux differences were observed in the production of steroids in M. furfur and in the metabolism of butanoate in M. pachydermatis. The predictions obtained via these metabolic reconstructions also suggested defects in the assimilation of palmitic acid in M. globosa, M. sympodialis, M. pachydermatis, and the atypical variant of M. furfur, but not in M. furfur. These predictions were validated via physiological characterization, showing the predictive power of metabolic network reconstructions to provide new clues about the metabolic versatility of Malassezia.

Compartmentalized metabolic network reconstruction of microbial communities to determine the effect of agricultural intervention on soils.

Soil microbial communities are responsible for a wide range of ecological processes and have an important economic impact in agriculture. Determining the metabolic processes performed by microbial communities is crucial for understanding and managing ecosystem properties. Metagenomic approaches allow the elucidation of the main metabolic processes that determine the performance of microbial communities under different environmental conditions and perturbations. Here we present the first compartmentalized metabolic reconstruction at a metagenomics scale of a microbial ecosystem. This systematic approach conceives a meta-organism without boundaries between individual organisms and allows the in silico evaluation of the effect of agricultural intervention on soils at a metagenomics level. To characterize the microbial ecosystems, topological properties, taxonomic and metabolic profiles, as well as a Flux Balance Analysis (FBA) were considered. Furthermore, topological and optimization algorithms were implemented to carry out the curation of the models, to ensure the continuity of the fluxes between the metabolic pathways, and to confirm the metabolite exchange between subcellular compartments. The proposed models provide specific information about ecosystems that are generally overlooked in non-compartmentalized or non-curated networks, like the influence of transport reactions in the metabolic processes, especially the important effect on mitochondrial processes, as well as provide more accurate results of the fluxes used to optimize the metabolic processes within the microbial community.

Metabolomic profile and nucleoside composition of Cordyceps nidus sp. nov. (Cordycipitaceae): A new source of active compounds.

Cordyceps sensu lato is a genus of arthropod-pathogenic fungi, which have been used traditionally as medicinal in Asia. Within the genus, Ophiocordyceps sinensis is the most coveted and expensive species in China. Nevertheless, harvesting wild specimens has become a challenge given that natural populations of the fungus are decreasing and because large-scale culture of it has not yet been achieved. The worldwide demand for products derived from cultivable fungal species with medicinal properties has increased recently. In this study, we propose a new species, Cordyceps nidus, which parasitizes underground nests of trapdoor spiders. This species is phylogenetically related to Cordyceps militaris, Cordyceps pruinosa, and a sibling species of Cordyceps caloceroides. It is found in tropical rainforests from Bolivia, Brazil, Colombia and Ecuador. We also investigated the medicinal potential of this fungus based on its biochemical properties when grown on four different culture media. The metabolic profile particularly that of nucleosides, in polar and non-polar extracts was determined by UPLC, and then correlated to their antimicrobial activity and total phenolic content. The metabolome showed a high and significant dependency on the substrate used for fungal growth. The mass intensities of nucleosides and derivative compounds were higher in natural culture media in comparison to artificial culture media. Among these compounds, cordycepin was the predominant, showing the potential use of this species as an alternative to O. sinensis. Furthermore, methanol fractions showed antimicrobial activity against gram-positive bacteria, and less than 3.00 mg of gallic acid equivalents per g of dried extract were obtained when assessing its total phenolic content by modified Folin-Ciocalteu method. The presence of polyphenols opens the possibility of further exploring the antioxidant capacity and the conditions that may enhance this characteristic. The metabolic composition and biochemical activity indicate potential use of C. nidus in pharmaceutical applications.

Defining the phylogenetic position of Amanita species from Andean Colombia.

Amanita is a worldwide-distributed fungal genus, with approximately 600 known species. Most species within the genus are ectomycorrhizal (ECM), with some saprotrophic representatives. In this study, we constructed the first comprehensive phylogeny including ECM species from Colombia collected in native Quercus humboldtii forests and in introduced Pinus patula plantations. We included 8 species (A. brunneolocularis, A. colombiana, A. flavoconia, A. fuligineodisca, A. muscaria, A. rubescens, A. sororcula, and A. xylinivolva) out of 16 species reported for the country, two new reports: A. citrina and A. virosa, and a new variety A. brunneolocularis var. pallida. Morphological taxonomic keys together with a phylogenetic approach using three nuclear gene regions: partial nuc rDNA 28S nuc rDNA internal transcribed spacers ITS1 and ITS2 and partial translation elongation factor 1-α gene (TEF1), were used to classify the specimens. Several highly supported clades were obtained from the phylogenetic hypotheses obtained by Bayesian inference and maximum likelihood approaches, allowing us to position the Colombian collections in a coherent infrageneric level and to contribute to the knowledge of local Amanita diversity.

Phytophthora infestans POPULATION STRUCTURE: A WORLDWIDE SCALE / Estructura poblacional de Phytophthora infestans: una escala global

Phytophthora infestans, the causal agent of late blight disease in potato and other members of the Solanaceae family, is responsible for causing the Irish potato famine and, even today, it causes enormous economic losses all over the world. For the establishment of an adequate pest management strategy, the determination of the pathogen's population structure is required. To characterize P. infestans populations worldwide two allozymes, Gpi (Glucose-6-phospate isomerase) and Pep ( Pep tidase), the RG57 DNA RFLP fingerprinting probe, as well as resistance to the fungicide metalaxyl and mating type, have been used as markers. P. infestans populations in Mexico have been one of the main focuses of research in the population biology of this pathogen because this country has been considered as one of the possible centers of origin of this oomycete. In this review we present the population structure of P. infestans in Mexico, Europe, Africa, Asia, North America, and South America, expanding it on the present situation of P. infestans in Colombia. Finally, we will discuss different lines of research that are being carried out today with respect to P. infestans in Colombia, which have shown the importance of continuing the study of this devastating plant pathogen in our country.

Phytophthora infestans, el agente causal del tizón tardío de la papa y otros miembros de la familia de las Solanáceas, es el responsable de la gran hambruna irlandesa y aún hoy sigue causando grandes pérdidas económicas alrededor del planeta. Para establecer estrategias de control adecuadas contra este patógeno se requiere comprender la estructura poblacional del mismo. Mundialmente se han utilizado como marcadores las aloenzimas, Gpi (Glucosa-6-fosfato isomerasa) y Pep (Peptidasa) y la sonda de fingerprinting de RFLP (Polimorfismos de la Longitud de los Fragmentos de Restricción), RG57. De igual forma, la resistencia al fungicida metalaxyl y el tipo de apareamiento, han sido empleados para caracterizar las poblaciones de P. infestans. Las poblaciones de P. infestans en México han sido uno de los focos principales de investigación en la biología poblacional de este patógeno debido a que este país ha sido considerado como uno de los posibles centros de origen de este oomiceto. En esta revisión se presentará la estructura poblacional de P. infestans en México, Europa, África, Asia, Norte América y Sur América, profundizando en la situación actual de P. infestans en Colombia. Finalmente, se discutirá las diferentes líneas de investigación que se llevan a cabo hoy respecto a P. infestans en Colombia, las cuales han mostrado la importancia de continuar con el estudio de este devastador patógeno de plantas en nuestro país.