Multiple Applications of Alamar Blue as an Indicator of Metabolic Function and Cellular Health in Cell Viability Bioassays

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Diversity and Oil Degradation Potential of Culturable Microbes Isolated from Chronically Contaminated Soils in Trinidad

Trinidad and Tobago is the largest producer of oil and natural gas in Central America and the Caribbean. Natural crude oil seeps, in addition to leaking petroleum pipelines, have resulted in chronic contamination of the surrounding terrestrial environments since the time of petroleum discovery, production, and refinement in Trinidad. In this study, we isolated microbes from soils chronically contaminated with crude oil using a culture-dependent approach with enrichment. The sampling of eight such sites located in the southern peninsula of Trinidad revealed a diverse microbial composition and novel oil-degrading filamentous fungi and yeast as single-isolate degraders and naturally occurring consortia, with specific bacterial species not previously reported in the literature. Multiple sequence comparisons and phylogenetic analyses confirmed the identity of the top degraders. The filamentous fungal community based on culturable species was dominated by Ascomycota, and the recovered yeast isolates were affiliated with Basidiomycota (65.23%) and Ascomycota (34.78%) phyla. Enhanced biodegradation of petroleum hydrocarbons is maintained by biocatalysts such as lipases. Five out of seven species demonstrated extracellular lipase activity in vitro. Our findings could provide new insights into microbial resources from chronically contaminated terrestrial environments, and this information will be beneficial to the bioremediation of petroleum contamination and other industrial applications

Supplementary Table S1 (a) and (b)

Supplementary table S1 (a) and (b) detailing the reference Internally transcribed spacer region (ITS) and beta-tubulin (BTUB) sequences of the five populations used in the stud

Fungicide Sensitivity among Isolates of Colletotrichum truncatum and Fusarium incarnatum-equiseti Species Complex Infecting Bell Pepper in Trinidad

Bell pepper is an economically important crop worldwide; however, production is restricted by a number of fungal diseases that cause significant yield loss. Chemical control is the most common approach adopted by growers to manage a number of these diseases. Monitoring for the development to resistance to fungicides in pathogenic fungal populations is central to devising integrated pest management strategies. Two fungal species, Fusarium incarnatum-equiseti species complex (FIESC) and Colletotrichum truncatum are important pathogens of bell pepper in Trinidad. This study was carried out to determine the sensitivity of 71 isolates belonging to these two fungal species to fungicides with different modes of action based on in vitro bioassays. There was no significant difference in log effective concentration required to achieve 50% colony growth inhibition (LogEC₅₀) values when field location and fungicide were considered for each species separately based on ANOVA analyses. However, the LogEC₅₀ value for the Aranguez-Antracol location-fungicide combination was almost twice the value for the Maloney/Macoya-Antracol location-fungicide combination regardless of fungal species. LogEC₅₀ values for Benomyl fungicide was also higher for C. truncatum isolates than for FIESC isolates and for any other fungicide. Cropping practices in these locations may explain the fungicide sensitivity data obtained

Selection of Fusarium Trichothecene Toxin Genes for Molecular Detection Depends on TRI Gene Cluster Organization and Gene Function

Food security is a global concern. Fusarium are among the most economically important fungal pathogens because they are ubiquitous, disease management remains a challenge, they produce mycotoxins that affect food and feed safety, and trichothecene mycotoxin production can increase the pathogenicity of some Fusarium species depending on the host species. Although trichothecenes may differ in structure by their patterns of hydroxylation or acetylation, these small changes have a significant impact on toxicity and the biological activity of these compounds. Therefore, detecting and identifying which chemotype is present in a given population are important to predicting the specific toxins that may be produced and, therefore, to evaluating the risk of exposure. Due to the challenges of inducing trichothecene production by Fusarium isolates in vitro for subsequent chemical analysis, PCR assays using gene-specific primers, either singly or in combination, designed against specific genes of the trichothecene gene cluster of multiple species of Fusarium have been developed. The establishment of TRI genotypes that potentially correspond to a specific chemotype requires examination of an information and knowledge pipeline whose critical aspects in sequential order are: (i) understanding the TRI gene cluster organization which differs according to Fusarium species under study; (ii) knowledge of the re-arrangements to the core TRI gene cluster over evolutionary time, which also differs according to Fusarium species; (iii) the functions of the TRI genes in the biosynthesis of trichothecene analogs; and (iv) based on (i)–(iii), selection of appropriate target TRI gene(s) for primer design in PCR amplification for the Fusarium species under study. This review, therefore, explains this pipeline and its connection to utilizing TRI genotypes as a possible proxy to chemotype designation

Fungicide Sensitivity among Isolates of Colletotrichum truncatum and Fusarium incarnatum-equiseti Species Complex Infecting Bell Pepper in Trinidad

Bell pepper is an economically important crop worldwide; however, production is restricted by a number of fungal diseases that cause significant yield loss. Chemical control is the most common approach adopted by growers to manage a number of these diseases. Monitoring for the development to resistance to fungicides in pathogenic fungal populations is central to devising integrated pest management strategies. Two fungal species, Fusarium incarnatum-equiseti species complex (FIESC) and Colletotrichum truncatum are important pathogens of bell pepper in Trinidad. This study was carried out to determine the sensitivity of 71 isolates belonging to these two fungal species to fungicides with different modes of action based on in vitro bioassays. There was no significant difference in log effective concentration required to achieve 50% colony growth inhibition (LogEC₅₀) values when field location and fungicide were considered for each species separately based on ANOVA analyses. However, the LogEC₅₀ value for the Aranguez-Antracol location-fungicide combination was almost twice the value for the Maloney/Macoya-Antracol location-fungicide combination regardless of fungal species. LogEC₅₀ values for Benomyl fungicide was also higher for C. truncatum isolates than for FIESC isolates and for any other fungicide. Cropping practices in these locations may explain the fungicide sensitivity data obtained

Comparative Sequence Analysis of TRI1 of Fusarium

Trichothecene mycotoxins are a class of secondary metabolites produced by multiple genera of fungi, including certain plant pathogenic Fusarium species. Functional variation in the TRI1 gene produces a novel Type A trichothecene called NX-2 in strains of F. graminearum. Using a bioinformatics approach, a systematic analysis of 52 translated TRI1 sequences of Fusarium species, including five F. graminearum NX-2 producers and four F. graminearum non-NX-2 producers, was conducted to explain the functional difference of TRI1p of FGNX-2. An assessment of several signature motifs of fungal P450s revealed amino acid substitutions in addition to the post-translational N-X-S/T sequons motif, which is indicative of N-linked glycosylation of this TRI1-encoded protein characteristic of NX-2 producers. There was evidence of selection bias, where TRI1 gene sequences were found to be under positive selection and, therefore, under functional constraints. The cumulative amino acid changes in the TRI1p sequences were reflected in the phylogenetic analyses which revealed species-specific clustering with a distinct separation of FGNX-2 from FG-non-NX-2 producers with high bootstrap support. Together, our findings provide insight into the amino acid sequence features responsible for the functional diversification of this TRI1p

TRI Genotyping and Chemotyping: A Balance of Power

Fusarium is among the top 10 most economically important plant pathogens in the world. Trichothecenes are the principal mycotoxins produced as secondary metabolites by select species of Fusarium and cause acute and chronic toxicity in animals and humans upon exposure either through consumption and/or contact. There are over 100 trichothecene metabolites and they can occur in a wide range of commodities that form food and feed products. This review discusses strategies to mitigate the risk of mycotoxin production and exposure by examining the Fusarium-trichothecene model. Fundamental to mitigation of risk is knowing the identity of the pathogen. As such, a comparison of current, recommended molecular approaches for sequence-based identification of Fusaria is presented, followed by an analysis of the rationale and methods of trichothecene (TRI) genotyping and chemotyping. This type of information confirms the source and nature of risk. While both are powerful tools for informing regulatory decisions, an assessment of the causes of incongruence between TRI genotyping and chemotyping data must be made. Reconciliation of this discordance will map the way forward in terms of optimization of molecular approaches, which includes data validation and sharing in the form of accessible repositories of genomic data and browsers for querying such data

Genetic structure and demographic history of Colletotrichum gloeosporioides sensu lato and C. truncatum isolates from Trinidad and Mexico

BACKGROUND: C. gloeosporioides sensu lato is one of the most economically important post-harvest diseases affecting papaya production worldwide. There is currently no information concerning the genetic structure or demographic history of this pathogen in any of the affected countries. Knowledge of molecular demographic parameters for different populations will improve our understanding of the biogeographic history as well as the evolutionary and adaptive potential of these pathogens. In this study, sequence data for ACT, GPDH, β-TUB and ITS gene regions were analyzed for C. gloeosporioides sensu lato and C. truncatum isolates infecting papaya in Trinidad and Mexico in order to determine the genetic structure and demographic history of these populations. RESULTS: The data indicated that Mexico is the ancestral C. gloeosporioides sensu lato population with asymmetrical migration to Trinidad. Mexico also had the larger effective population size but, both Mexico and Trinidad populations exhibited population expansion. Mexico also had greater nucleotide diversity and high levels of diversity for each gene. There was significant sub-division of the Trinidad and Mexico populations and low levels of genetic divergence among populations for three of the four gene regions; β-TUB was shown to be under positive selection. There were also dissimilar haplotype characteristics for both populations. Mutation may play a role in shaping the population structure of C. gloeosporioides sensu lato isolates from Trinidad and from Mexico, especially with respect to the ACT and GPDH gene regions. There was no evidence of gene flow between the C. truncatum populations and it is possible that the Mexico and Trinidad populations emerged independently of each other. CONCLUSIONS: The study revealed relevant information based on the genetic structure as well as the demographic history of two fungal pathogens infecting papaya, C. gloeosporioides sensu lato and C. truncatum, in Trinidad and Mexico. Understanding the genetic structure of pathogen populations will assist in determining the evolutionary potential of the pathogen and in identifying which evolutionary forces may have the greatest impact on durability of resistance. Intervention strategies that target these evolutionary forces would prove to be the most practical