Detection and diversity of the mannosylerythritol lipid (MEL) gene cluster and lipase A and B genes of Moesziomyces antarcticus isolated from terrestrial sites chronically contaminated with crude oil in Trinidad.

BACKGROUND: Mannosylerythritol lipids (MELs) belong to the class of glycolipid biosurfactants and are produced by members of the Ustilago and Moesziomyces genera. Production of MELs is regulated by a biosynthetic gene cluster (MEL BGC). Extracellular lipase activity is also associated with MEL production. Most microbial glycolipid-producers are isolated from oil-contaminated environments. MEL-producing yeast that are capable of metabolizing crude oil are understudied, and there is very limited data on indigenous strains from tropical climates. Analysis of the MEL BGC and lipase genes in Trinidad M. antarcticus strains, using a gene-targeted approach, revealed a correlation between their intrinsic capability to degrade crude oil and their adaptation to survive in a chronically polluted terrestrial environment. RESULTS: M. antarcticus was isolated from naturally-occurring crude oil seeps and an asphaltic mud volcano in Trinidad; these are habitats that have not been previously reported for this species. Genus identification was confirmed by the large-subunit (LSU) and the small-subunit (SSU) sequence comparisons and species identification was confirmed by ITS sequence comparisons and phylogenetic inference. The essential genes (Emt1, Mac1, Mac2, Mmf1) of the MEL BGC were detected with gene-specific primers. Emt1p, Mac1p and Mmf1p sequence analyses confirmed that the Trinidad strains harboured novel synonymous amino acid (aa) substitutions and structural comparisons revealed different regions of disorder, specifically for the Emt1p sequence. Functionality of each protein sequence was confirmed through motif mining and mutation prediction. Phylogenetic relatedness was inferred for Emt1p, Mac1p and Mmf1p sequences. The Trinidad strains clustered with other M. antarcticus sequences, however, the representative Trinidad M. antarcticus sequences consistently formed a separate, highly supported branch for each protein. Similar phylogenetic placement was indicated for LipA and LipB nucleotide and protein sequences. The Trinidad strains also demonstrated lipolytic activity in culture, with an ability to utilize different carbon sources. Comparative evolution of MEL BGC and LipA gene suggested early and late duplication events, depending on the gene, followed by a number of speciation events within Ustilaginaceae. M. antarcticus and M. aphidis were separated from all other members of Ustilaginaceae and two gene homologues were detected, one for each species. CONCLUSIONS: Sequence analyses was based on a novel gene-targeted approach to analyze the essential genes of the MEL BGC and LipA and LipB genes of M. antarcticus strains from Trinidad. The findings indicated that these strains accumulated nucleotide mutations to a threshold level that did not affect the function of specific proteins encoded by the MEL BGC and LipA and LipB genes. The biosurfactant and lipase enzymes secreted by these Trinidad M. antarcticus strains facilitated their survival in oil-contaminated terrestrial environments. These findings suggest that the Trinidad strains should be explored as promising candidates for the commercial production of MEL biosurfactants and lipase enzymes.

Molecular signatures of Janthinobacterium lividum from Trinidad support high potential for crude oil metabolism.

BACKGROUND: Janthinobacterium lividum is considered to be a psychrotrophic bacterial species. For the first time in the literature, J. lividum strains were isolated from Trinidad presenting with atypical features - hydrocarbonoclastic and able to survive in a tropical environment. METHODS: Identification of the Trinidad strains was carried out through 16S rRNA phylogenetic analysis. Gene-specific primers were designed to target the VioA which encodes violacein pigment and the EstA/B gene which encodes secreted extracellular lipase. Bioinformatics analyses were carried out on the nucleotide and amino acid sequences of VioA and EstA/B genes of the Trinidad Janthinobacterium strains to assess functionality and phylogenetic relatedness to other Janthinobacterium sequences specifically and more broadly, to other members of the Oxalobacteraceae family of betaproteobacteria. RESULTS: 16S rRNA confirmed the identity of the Trinidad strains as J. lividum and resolved three of the Trinidad strains at the intra-specific level. Typical motility patterns of this species were recorded. VioAp sequences were highly conserved, however, synonymous substitutions located outside of the critical sites for enzyme function were detected for the Trinidad strains. Comparisons with PDB 6g2p model from aa231 to aa406 further indicated no functional disruption of the VioA gene of the Trinidad strains. Phylogeny of the VioA protein sequences inferred placement of all J. lividum taxa into a highly supported species-specific clade (bs = 98%). EstA/Bp sequences were highly conserved, however, synonymous substitutions were detected that were unique to the Trinidad strains. Phylogenetic inference positioned the Trinidad consensus VioA and EstA protein sequences in a clearly distinct branch. CONCLUSIONS: The findings showed that the primary sequence of VioAp and EstA/Bp were unique to the Trinidad strains and these molecular signatures were reflected in phylogenetic inference. Our results supported chemotaxis, possible elective inactivation of VioA gene expression and secreted lipase activity as survival mechanisms of the Trinidad strains in petrogenic conditions.

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, ß-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; ß-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.

Genetic structure of Colletotrichum gloeosporioides sensu lato isolates infecting papaya inferred by multilocus ISSR markers.

Colletotrichum gloeosporioides sensu lato is widely distributed throughout temperate and tropical regions and causes anthracnose disease in numerous plant species. Development of effective disease management strategies is dependent on, among other factors, an understanding of pathogen genetic diversity and population stratification at the intraspecific level. For 132 isolates of C. gloeosporioides sensu lato collected from papaya in Trinidad, inter-simple-sequence repeat-polymerase chain reaction (ISSR-PCR) generated 121 polymorphic loci from five ISSR primers selected from an initial screen of 22 ISSR primers. The mean percentage of polymorphic loci was 99.18%. Bayesian cluster analysis inferred three genetic subpopulations, where group 1 consisted exclusively of isolates collected in the southern part of Trinidad whereas groups 2 and 3, although genetically distinct, were mixtures of isolates collected from both the northern and southern parts of Trinidad. Principal coordinates analysis and unweighted pair-group method with arithmetic mean phylogeny were concordant with Bayesian cluster analysis and supported subdivision into the three subpopulations. Overall, the total mean gene diversity was 0.279, the mean within-population gene diversity was 0.2161, and genetic differentiation for the Trinidad population was 0.225. Regionally, northern isolates had a lower gene diversity compared with southern isolates. Nei's gene diversity was highest for group 1 (h = 0.231), followed by group 2 (h = 0.215) and group 3 (h = 0.202). Genotypic diversity was at or near maximum for all three subpopulations after clone correction. Pairwise estimates of differentiation indicated high and significant genetic differentiation among the inferred subpopulations (Weir's θ of 0.212 to 0.325). Pairwise comparisons among subpopulations suggested restricted gene flow between groups 1 and 2 and groups 1 and 3 but not between groups 2 and 3. The null hypothesis of random mating was rejected for all three inferred subpopulations. These results suggest that pathogen biology and epidemiology as well as certain evolutionary factors may play an important role in population substructuring of C. gloeosporioides sensu lato isolates infecting papaya in Trinidad.

Differential Responses of Colletotrichum gloeosporioides and C. truncatum Isolates from Different Hosts to Multiple Fungicides Based on Two Assays.

Anthracnose is one of the most important postharvest diseases of many economically important crops worldwide. This study was conducted with the objective of investigating the sensitivity of Colletotrichum gloeosporioides and C. truncatum isolates to multiple fungicides with different modes of action. The study analyzed quantitative sensitivity data derived from conventional amended agar (AA) assays and qualitative spore responses obtained from a novel microtiter bioassay that is based on reduction of a viability dye, Alamar blue (AB). Generally, for AA assays, the percent growth inhibition (%RGI) increased with increasing concentration for all isolates and all fungicides, except for copper hydroxide. C. truncatum isolates reacted differently to increasing concentrations of the various fungicides depending on whether the isolates originated from pepper or papaya. C. truncatum from pepper had generally less %RGI than C. truncatum isolates from papaya. C. gloeosporioides isolates from papaya had generally higher %RGI than C. truncatum isolates for all concentrations tested for pyraclostrobin, chlorothalonil, and fosetyl-aluminum. C. gloeosporioides isolates from pepper had generally higher %RGI than C. truncatum isolates for all concentrations tested for most fungicides. In all cases, Colletotrichum sp. and fungicide had significant (P ≤ 0.001) effects on the log concentration of fungicide for which relative growth was inhibited by 50 and 90% (log EC50 and log EC90, respectively) calculated for all isolates, regardless of whether values were compared for only C. gloeosporioides isolates or only C. truncatum isolates. Correlation analyses of log EC50 and log EC90 values of all the isolates revealed a nonsignificant association for pyraclostrobin. In AB assays, all fungicides had an equivalent effect at inhibiting spore germination at the lower concentrations. According to binary logistic regression analyses, species, isolate, and fungicide concentration had significant predictive value in determining whether an AB test would be positive. Sequence alignments between C. gloeosporioides isolates and C. gloeosporioides f. sp. aeschynomene revealed no base substitutions at codons 198, 199, 200, and 240; however, sequence comparisons between C. truncatum isolates and C. gloeosporioides f. sp. aeschynomene revealed two codon changes located outside of the identified codon 198 or 200 associated with the benzimidazole-resistant phenotype of C. gloeosporioides isolates.

Multiple applications of Alamar Blue as an indicator of metabolic function and cellular health in cell viability bioassays.

Accurate prediction of the adverse effects of test compounds on living systems, detection of toxic thresholds, and expansion of experimental data sets to include multiple toxicity end-point analysis are required for any robust screening regime. Alamar Blue is an important redox indicator that is used to evaluate metabolic function and cellular health. The Alamar Blue bioassay has been utilized over the past 50 years to assess cell viability and cytotoxicity in a range of biological and environmental systems and in a number of cell types including bacteria, yeast, fungi, protozoa and cultured mammalian and piscine cells. It offers several advantages over other metabolic indicators and other cytotoxicity assays. However, as with any bioassay, suitability must be determined for each application and cell model. This review seeks to highlight many of the important considerations involved in assay use and design in addition to the potential pitfalls.

A Rapid Colorimetric Microtiter Bioassay to Evaluate Fungicide Sensitivity Among Verticillium dahliae Isolates.

Management of Verticillium wilt relies on the adoption of integrated control strategies. The effects of long-term chemical use may be negated by the development of fungicide resistance. A rapid colorimetric bioassay was developed to evaluate sensitivity of V. dahliae isolates to differently acting fungicides. This assay capitalizes on the advantages of a 96-well microtiter plate format and the nontoxic cell viability dye, Alamar Blue (AB). Analysis of variance revealed that incubation time, spore density, and media type were important parameters that must be optimized for the AB assay. The effective linear range of the assay was dependent on incubation time and spore density. Survival of 107 spores/ml of each of 10 isolates in the presence of a range of serial dilutions of nine commercial fungicides was assessed. Effective concentrations at 50 and 90% inhibition of growth were calculated for each fungicide. A comparison of the percent growth inhibition for each fungicide at 0.6 mg/ml, as determined by AB and amended-agar assays, revealed a strong positive correlation for six of the fungicides. The optimized AB assay proved to be a rapid and reproducible method of testing the efficacy of fungicides with the option of deriving quantitative or qualitative data.

Molecular and Phenotypic Characterization of Colletotrichum Species Associated with Anthracnose Disease of Papaya in Trinidad.

Anthracnose disease is a major limiting factor to papaya production worldwide. Accurate identification of the pathogens responsible for this disease is important to developing disease management strategies. One hundred and three (103) isolates of Colletotrichum were collected from infected papaya fruits cvs. Red lady and Tainung No. 2 - F1 hybrid in Trinidad. Of all isolates, 79% were C. gloeosporioides and 21% were C. truncatum. Spore morphology, cultural characteristics, differential reaction to benomyl in addition to ITS1 and ß-tubulin gene sequence comparisons unequivocally identified and separated the two species of Colletotrichum. Certain characteristics enabled discrimination between the two species and may be used for provisional identification of these species isolated from papaya. Isolates of C. gloeosporioides grew at a significantly faster rate than those of C. truncatum. C. gloeosporioides isolates were sensitive to benomyl at 1.0 µg/ml, but C. truncatum isolates were resistant. Pathogenicity tests revealed that Colletotrichum species and papaya cultivar had no significant effect on lesion diameter. In cross-infection studies, isolates of C. gloeosporioides from mango and C. truncatum from sweet pepper were able to infect and cause symptoms in wounded mature papaya fruit under controlled conditions. There was no evidence of anthracnose infection in seeds of infected fruits based on the results of growing-on tests. Phylogenetic analyses were based on comparisons of ITS1 and ß-tubulin gene sequences. Both neighbor-joining and maximum parsimony methods resolved all Colletotrichum isolates from papaya into species-specific clusters with high bootstrap support. Additionally, a pair of species-specific primers were developed (Ct-TUB-F/R) which allowed reliable detection of C. truncatum isolates from papaya.

Spatial pattern of genetic diversity in field populations of Fusarium incarnatum-equiseti species complex.

Fusarium is associated with a number of wilt, blight, scab, and rot diseases in a range of economically important staple food crops worldwide. An assessment of the genetic structure and population stratification of Fusarium incarnatum-equiseti species complex (FIESC) pathogen populations is important to understand the evolutionary potential of such populations in adapting to environmental change. Based on intersimple sequence repeat polymerase chain reaction (ISSR-PCR), it was found that the pathogen population was structured into three genetic clusters for which genetic differentiation was higher within than among populations. There was high intrapopulation genetic diversity for population 1 (94.63%) which consisted largely of isolates collected from North Trinidad. Populations 2 and 3 had a low level of admixture among the populations based on overall population differentiation. Population 1 accounted for the highest amount of genetic variation (95.82%) followed by populations 2 and 3. Population stratification was reflected in the dendrogram topology, which consisted of three main genetic clusters and which coincided with the outcome of Bayesian and PCoA analyses. The populations were isolated by distance, and Voronoi tessellations indicated physical or structural barriers to gene flow which contributed to restricted admixture between two of three populations. These findings suggest a high evolutionary potential for this FIESC pathogen population, the implications of which directly affect disease management strategies.

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.

Biodiversity and biocatalyst activity of culturable hydrocarbonoclastic fungi isolated from Marac-Moruga mud volcano in South Trinidad.

Mud volcanoes (MVs) are visible signs of oil and gas reserves present deep beneath land and sea. The Marac MV in Trinidad is the only MV associated with natural hydrocarbon seeps. Petrogenic polyaromatic hydrocarbons (PAHs) in its sediments must undergo biogeochemical cycles of detoxification as they can enter the water table and aquifers threatening ecosystems and biota. Recurrent hydrocarbon seep activity of MVs consolidates the growth of hydrocarbonoclastic fungal communities. Fungi possess advantageous metabolic and ecophysiological features for remediation but are underexplored compared to bacteria. Additionally, indigenous fungi are more efficient at PAH detoxification than commercial/foreign counterparts and remediation strategies remain site-specific. Few studies have focused on hydrocarbonoclastic fungal incidence and potential in MVs, an aspect that has not been explored in Trinidad. This study determined the unique biodiversity of culturable fungi from the Marac MV capable of metabolizing PAHs in vitro and investigated their extracellular peroxidase activity to utilize different substrates ergo their extracellular oxidoreductase activity (> 50% of the strains decolourized of methylene blue dye). Dothideomycetes and Eurotiomycetes (89% combined incidence) were predominantly isolated. ITS rDNA sequence cluster analysis confirmed strain identities. 18 indigenous hydrocarbonoclastic strains not previously reported in the literature and some of which were biosurfactant-producing, were identified. Intra-strain variability was apparent for PAH utilization, oil-tolerance and hydroxylase substrate specificity. Comparatively high levels of extracellular protein were detected for strains that demonstrated low substrate specificity. Halotolerant strains were also recovered which indicated marine-mixed substrata of the MV as a result of deep sea conduits. This work highlighted novel MV fungal strains as potential bioremediators and biocatalysts with a broad industrial applications.

Pathogenomics and Management of Fusarium Diseases in Plants.

There is an urgency to supplant the heavy reliance on chemical control of Fusarium diseases in different economically important, staple food crops due to development of resistance in the pathogen population, the high cost of production to the risk-averse grower, and the concomitant environmental impacts. Pathogenomics has enabled (i) the creation of genetic inventories which identify those putative genes, regulators, and effectors that are associated with virulence, pathogenicity, and primary and secondary metabolism; (ii) comparison of such genes among related pathogens; (iii) identification of potential genetic targets for chemical control; and (iv) better characterization of the complex dynamics of host-microbe interactions that lead to disease. This type of genomic data serves to inform host-induced gene silencing (HIGS) technology for targeted disruption of transcription of select genes for the control of Fusarium diseases. This review discusses the various repositories and browser access points for comparison of genomic data, the strategies for identification and selection of pathogenicity- and virulence-associated genes and effectors in different Fusarium species, HIGS and successful Fusarium disease control trials with a consideration of loss of RNAi, off-target effects, and future challenges in applying HIGS for management of Fusarium diseases.

Three-Locus Sequence Identification and Differential Tebuconazole Sensitivity Suggest Novel Fusarium equiseti Haplotype from Trinidad.

The Fusarium incarnatum-equiseti species complex (FIESC) consists of 33 phylogenetic species according to multi-locus sequence typing (MLST) and Genealogical Concordance Phylogenetic Species Recognition (GCPSR). A multi-locus dataset consisting of nucleotide sequences of the translation elongation factor (EF-1α), calmodulin (CAM), partial RNA polymerase largest subunit (RPB1), and partial RNA polymerase second largest subunit (RPB2), was generated to distinguish among phylogenetic species within the FIESC isolates infecting bell pepper in Trinidad. Three phylogenetic species belonged to the Incarnatum clade (FIESC-15, FIESC-16, and FIESC-26), and one species belonged to the Equiseti clade (FIESC-14). Specific MLST types were sensitive to 10 µg/mL of tebuconazole fungicide as a discriminatory dose. The EC50 values were significantly different among the four MLST groups, which were separated into two homogeneous groups: FIESC-26a and FIESC-14a, demonstrating the "sensitive" azole phenotype and FIESC-15a and FIESC-16a as the "less sensitive" azole phenotype. CYP51C sequences of the Trinidad isolates, although under positive selection, were without any signatures of recombination, were highly conserved, and were not correlated with these azole phenotypes. CYP51C sequences were unable to resolve the FIESC isolates as phylogenetic inference indicated polytomic branching for these sequences. This data is important to different research communities, including those studying Fusarium phytopathology, mycotoxins, and public health impacts.

Diversity, structure, and synteny of the cutinase gene of Colletotrichum species.

Colletotrichum species complexes are among the top 10 economically important fungal plant pathogens worldwide because they can infect climacteric and nonclimacteric fruit at the pre and/or postharvest stages. C. truncatum is the major pathogen responsible for anthracnose of green and red bell pepper fruit worldwide. C. brevisporum was recently reported to be a minor pathogen of red bell pepper fruit in Trinidad, but has recently been reported as pathogenic to other host species in other countries. The ability of these phytopathogens to produce and secrete cutinase is required for dismantling the cuticle of the host plant and, therefore, crucial to the necrotrophic phase of their infection strategy. In vitro bioassays using different lipid substrates confirmed the ability of C. truncatum and C. brevisporum isolates from green and red bell peppers to secrete cutinase. The diversity, structure and organization and synteny of the cutinase gene were determined among different Colletotrichum species. Cluster analysis indicated a low level of nucleotide variation among C. truncatum sequences. Nucleotide sequences of C. brevisporum were more related to C. truncatum cutinase nucleotide sequences than to C. gloeosporioides. Cluster patterns coincided with haplotype and there was evidence of significant positive selection with no recombination signatures. The structure of the cutinase gene included two exons with one intervening intron and, therefore, one splice variant. Although amino acid sequences were highly conserved among C. truncatum isolates, diversity "hot spots" were revealed when the 66-amino acid coding region of 200 fungal species was compared. Twenty cutinase orthologues were detected among different fungal species, whose common ancestor is Pezizomycotina and it is purported that these orthologues arose through a single gene duplication event prior to speciation. The cutinase domain was retained both in structure and arrangement among 34 different Colletotrichum species. The order of aligned genomic blocks between species and the arrangement of flanking protein domains were also conserved and shared for those domains immediately located at the N- and C-terminus of the cutinase domain. Among these were an RNA recognition motif, translation elongation factor, signal peptide, pentatricopeptide repeat, and Hsp70 family of chaperone proteins, all of which support the expression of the cutinase gene. The findings of this study are important to understanding the evolution of the cutinase gene in C. truncatum as a key component of the biotrophic-necrotrophic switch which may be useful in developing gene-targeting strategies to decrease the pathogenic potential of Colletotrichum species.

Signatures of TRI5, TRI8 and TRI11 Protein Sequences of Fusarium incarnatum-equiseti Species Complex (FIESC) Indicate Differential Trichothecene Analogue Production.

The variability and phylogeny among TRI5, TRI8 and TRI11 nucleotide and translated protein sequences of isolates from Trinidad belonging to Fusarium incarnatum-equiseti species complex (FIESC) were compared with FIESC reference sequences. Taxa appeared to be more divergent when DNA sequences were analyzed compared to protein sequences. Neutral and non-neutral mutations in TRI protein sequences that may correspond to variability in the function and structure of the selected TRI proteins were identified. TRI5p had the lowest amino acid diversity with zero predicted non-neutral mutations. TRI5p had potentially three protein disorder regions compared to TRI8p with five protein disorder regions. The deduced TRI11p was more conserved than TRI8p of the same strains. Amino acid substitutions that may be non-neutral to protein function were only detected in diacetoxyscirpenol (DAS) and fusarenon-X (FUS-X) producers of the reference sequence subset for TRI8p and TRI11p. The deduced TRI5 and TRI8 amino acid sequences were mapped to known 3D-structure models and indicated that variations in specific protein order/disorder regions exist in these sequences which affect the overall structural conservation of TRI proteins. Assigning single or combination non-neutral mutations to a particular toxicogenic phenotype may be more representative of potential compared to using genotypic data alone, especially in the absence of wet-lab, experimental validation.

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.

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.

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.

Utility of DNA barcoding to identify rare endemic vascular plant species in Trinidad.

The islands of the Caribbean are considered to be a "biodiversity hotspot." Collectively, a high level of endemism for several plant groups has been reported for this region. Biodiversity conservation should, in part, be informed by taxonomy, population status, and distribution of flora. One taxonomic impediment to species inventory and management is correct identification as conventional morphology-based assessment is subject to several caveats. DNA barcoding can be a useful tool to quickly and accurately identify species and has the potential to prompt the discovery of new species. In this study, the ability of DNA barcoding to confirm the identities of 14 endangered endemic vascular plant species in Trinidad was assessed using three DNA barcodes (matK, rbcL, and rpoC1). Herbarium identifications were previously made for all species under study. matK, rbcL, and rpoC1 markers were successful in amplifying target regions for seven of the 14 species. rpoC1 sequences required extensive editing and were unusable. rbcL primers resulted in cleanest reads, however, matK appeared to be superior to rbcL based on a number of parameters assessed including level of DNA polymorphism in the sequences, genetic distance, reference library coverage based on BLASTN statistics, direct sequence comparisons within "best match" and "best close match" criteria, and finally, degree of clustering with moderate to strong bootstrap support (>60%) in neighbor-joining tree-based comparisons. The performance of both markers seemed to be species-specific based on the parameters examined. Overall, the Trinidad sequences were accurately identified to the genus level for all endemic plant species successfully amplified and sequenced using both matK and rbcL markers. DNA barcoding can contribute to taxonomic and biodiversity research and will complement efforts to select taxa for various molecular ecology and population genetics studies.