Reference genes for Eucalyptus spp. under Beauveria bassiana inoculation and subsequently infestation by the galling wasp Leptocybe invasa.

Relative gene expression analysis through RT-qPCR is an important molecular technique that helps understanding different molecular mechanisms, such as the plant defense response to insect pests. However, the use of RT-qPCR for gene expression analysis can be affected by factors that directly affect the reliability of the results. Among these factors, the appropriate choice of reference genes is crucial and can strongly impact RT-qPCR relative gene expression analyses, highlighting the importance in correctly choosing the most suitable genes for the success of the analysis. Thus, this study aimed to select and validate reference genes for relative gene expression studies through RT-qPCR in hybrids of Eucalyptus tereticornis × Eucalyptus camaldulensis (drought tolerant and susceptible to Leptocybe invasa) under conditions of inoculation by the Beauveria bassiana fungus and subsequent infestation by L. invasa. The expression level and stability of eleven candidate genes were evaluated. Stability was analyzed using the RefFinder tool, which integrates the geNorm, NormFinder, BestKeeper, and Delta-Ct algorithms. The selected reference genes were validated through the expression analysis of the transcriptional factor EcDREB2 (dehydration-responsive element-binding protein 2). For all treatments evaluated, EcPTB, EcPP2A-1, and EcEUC12 were the best reference genes. The triplets EcPTB/EcEUC12/EcUBP6, EcPP2A-1/EcEUC12/EcPTB, EcIDH/EcSAND/Ecα-TUB, EcPP2A-1/Ecα-TUB/EcPTB, and EcPP2A-1/EcUPL7/EcSAND were the best reference genes for the control plants, mother plants, plants inoculated with B. bassiana, plants infested with L. invasa, and plants inoculated with B. bassiana and subsequently infested with L. invasa, respectively. The best determined reference genes were used to normalize the RT-qPCR expression data for each experimental condition evaluated. The results emphasize the importance of this type of study to ensure the reliability of relative gene expression analyses. Furthermore, the findings of this study can be used as a basis for future research, comprising gene expression analysis of different eucalyptus metabolic pathways.

Selection and validation of reference genes for RT-qPCR gene expression studies in Candida viswanathii cultivated under different grown conditions.

The properties presented by Candida viswanathii's lipases turn this specie into a promising producer of potentially applicable lipases in several industrial sectors, such as: food, textiles, in the oleochemical and paper industries, and also in different pharmaceutical applications. However, studies for elucidating growth and developmental processes at the molecular level in this species are still incipient. Performing such kinds of studies often rely on the use of the RT-qPCR, which is a highly sensitivity technique, but whose parameters must be carefully planned for achieving reliable data. Among the crucial parameters required for achieving reliable results through this technique, the use of appropriated and validated reference genes is one the most important, constituting a bottleneck, mainly in species where molecular studies are scarce. Thus, the aim of this study was to determine the best reference genes for RT-qPCR gene expression studies in C. viswanathii grown in culture media containing four different carbon sources (Olive oil, Triolein, Tributyrin, and Glucose). Eleven candidate reference genes (ACT, GPH1, AGL9, RPB2, SAP1, PGK1, TAF10, UBC13, TFC1, UBP6, and FBA1) were analyzed for their expression patterns and stability. Analysis of gene expression stability was performed using the RefFinder tool, which integrates the geNorm, NormFinder, BestKeeper and Delta-Ct algorithms, and validation of the results was performed through analyzing the expression of a lipase gene, CvLIP4. Analyzing the four treatments together, CvACT and CvRPB2 constituted the best reference gene pair. When treatments are analyzed individually, CvRPB2/CvACT, CvFBA1/CvAGL9, CvPGK1/CvAGL9 and CvACT/CvRPB2 were the best reference gene pairs for the culture media containing olive oil, triolein, tributyrin, and glucose as carbon sources, respectively. These results are essential and form the basis for the development of relative gene expression studies in C. viswanathii, since adequate reference genes are crucial for the reliability of RT-qPCR data.

Comparative assessment of three RNA extraction methods for obtaining high-quality RNA from Candida viswanathii biomass.

Isolating high quality RNA is a limiting factor in molecular analysis, since it is the base for transcriptional studies. The RNA extraction method can directly affect the RNA quality and quantity, as well as, its overall cost. The industrial importance of the yeast genus Candida in several sectors comes from their capacity to produce Lipases. These enzymes are one of the main metabolites produced by some Candida species, and it has been shown that Candida yeast can biodegrade petroleum hydrocarbons and diesel oil from biosurfactants that they can produce, a feature that turns these organisms into potential combatants for bioremediation techniques. Thus, this study aimed to determine an efficient method for isolating high quality RNA from Candida viswanathii biomass. To achieve this aim, three different RNA extraction methods, TRIzol, Hot Acid Phenol, and CTAB (Cetyltrimethylammonium Bromide), were tested. The three tested methods allowed the isolation of high-quality RNA from C. viswanathii biomass and yielded suitable RNA quantity for carrying out RT-qPCR studies. In addition, all methods displayed high sensitivity for the expression analysis of the CvGPH1 gene through RT-qPCR, with TRIzol and CTAB showing the best results and the CTAB method displaying the best cost-benefit ratio (US$0.35/sample).

Diagnosing the novel SARS-CoV-2 by quantitative RT-PCR: variations and opportunities.

The world is currently facing a novel viral pandemic (SARS-CoV-2), and large-scale testing is central to decision-making for the design of effective policies and control strategies to minimize its impact on the global population. However, testing for the presence of the virus is a major bottleneck in tracking the spreading of the disease. Given its adaptability regarding the nucleotide sequence of target regions, RT-qPCR is a strong ally to reveal the rapid geographical spreading of novel viruses. We assessed PCR variations in the SARS-CoV-2 diagnosis taking into account public genome sequences and diagnosis kits used by different countries. We analyzed 226 SARS-CoV-2 genome sequences from samples collected by March 22, 2020. Our work utilizes a phylogenetic approach that reveals the early evolution of the virus sequence as it spreads around the globe and informs the design of RT-qPCR primers and probes. The quick expansion of testing capabilities of a country during a pandemic is largely impaired by the availability of adequately trained personnel on RNA isolation and PCR analysis, as well as the availability of hardware (thermocyclers). We propose that rapid capacity development can circumvent these bottlenecks by training medical and non-medical personnel with some laboratory experience, such as biology-related graduate students. Furthermore, the use of thermocyclers available in academic and commercial labs can be promptly calibrated and certified to properly conduct testing during a pandemic. A decentralized, fast-acting training and testing certification pipeline will better prepare us to manage future pandemics.

Early histological, hormonal, and molecular changes during pineapple (Ananas comosus (L.) Merrill) artificial flowering induction.

Natural flowering can cause serious scheduling problems in the pineapple (Ananas comosus) industry and increase harvest costs. Pineapple flowering is thought to be triggered by increased ethylene levels and artificial forcing of pineapple flowering is a common practice to promote flowering synchronisation. However, little is known about the early hormonal and molecular changes of pineapple flowering induction and development. Here, we aimed to analyse the molecular, hormonal, and histological changes during artificial pineapple flowering by Ethrel®48 treatment. Histological analyses of the shoot apical meristem, leaf gibberellic acid (GA3), and ethylene quantification were carried out during the first 72h after Ethrel®48 treatment. Expression profiles from ethylene biosynthesis (AcACS2 and AcACO1), gibberellin metabolism (AcGA2-ox1 and AcDELLA1), and flower development (FT-like gene (AcFT), LFY-like gene (AcLFY), and a PISTILLATA-like gene (AcPI)) genes were analysed during the first 24h after Ethrel®48 treatment. Differentiation processes of the shoot apical meristem into flower buds were already present in the first 72h after Ethrel®48 treatment. Ethrel®48 lead to a reduction in GA3 levels, probably triggered by elevated ethylene levels and the positive regulation AcGA2-ox1. AcLFY activation upon Ethrel®48 may also have contributed to the reduction of GA3 levels and, along with the up-regulation of AcPI, are probably associated with the flower induction activation. AcFT and AcDELLA1 do not seem to be regulated by GA3 and ethylene. Decreased GA3 and increased ethylene levels suggest an accumulation of AcDELLA1, which may display an important role in pineapple flowering induction. Thus, this study shows that molecular, hormonal, and histological changes are present right after Ethrel®48 treatment, providing new insights into how pineapple flowering occurs under natural conditions.

Tipos de estacas e diferentes substratos na propagação vegetativa da erva cidreira (quimiotipos I, II e III) / Types of cuttings and different substrates in the vegetative propagation of the Lippia alba (chemotypes I, II and III)

A erva-cidreira [Lippia alba (Mill.) N. E. Brown] é comumente utilizada no Brasil como planta medicinal por suas propriedades analgésica, antiespasmódica, calmante e sedativa. O objetivo do trabalho foi avaliar o efeito do tipo de estaca e de diferentes substratos sobre a propagação vegetativa da erva-cidreira quimiotipos I, II e III. O material botânico utilizado foi estacas de erva-cidreira quimiotipos I, II, e III, com aproximadamente 12 cm de comprimento. No primeiro experimento, foram utilizadas estacas (apicais e basais) dos três quimiotipos, em um esquema fatorial 2 x 3. No segundo experimento, as estacas dos três quimiotipos foram propagadas em três substratos: substrato comercial + esterco bovino curtido (na proporção de 1:1); substrato comercial + palha de arroz carbonizada (na proporção de 1:1); e substrato comercial, em um esquema fatorial de 3 x 3 com 4 repetições, onde se determinou a massa fresca e seca da raiz, caule e folhas. Estacas apicais e basais podem ser utilizadas na propagação vegetativa da erva-cidreira quimiotipos I, II e III. As misturas substrato comercial + palha de arroz carbonizada e substrato comercial + esterco bovino curtido são recomendadas para a propagação vegetativa da erva-cidreira quimiotipos I, II e III.

The Lippia alba is commonly used in Brazil as a medicinal plant for its analgesic properties, antispasmodic, calming and sedative. The objective of this study was to evaluate the effect of cutting type and different substrates on the propagation of the Lippia alba chemotypes I, II and III. The botanical material used was stakes Lippia alba chemotypes I, II and III, with approximately 12 cm in length. In the first experiment, we used cuttings (apical and basal) of the three chemotypes in a 2 x 3 factorial arrangement. In the second experiment, cuttings of three chemotypes were propagated in three substrates: commercial substrate + cattle manure (1:1), commercial substrate + carbonized rice hulls (1:1) and commercial substrate, in a factorial 3 x 3 with 4 replicates, was determined fresh and dry weight of root, stem and leaves. Apical and basal cuttings can be used in the propagation of the Lippia alba chemotypes I, II and III. The commercial substrate mixtures + carbonized rice hulls and commercial substrate + cattle manure are recommended for the propagation of lemongrass chemotypes I, II and III.

Estabilidade e adaptabilidade da produtividade e da reação a insetos de solo em genótipos experimentais e comerciais de batata-doce / Stability and adaptability in the yield and reaction to soil insects in commercial and experimental genotypes of sweet potato

O objetivo desse trabalho foi estimar a adaptabilidade e estabilidade de genótipos batata-doce em três ambientes na região Centro-Sul do Estado do Tocantins. O delineamento experimental utilizado foi o de blocos ao acaso com três repetições e dezoito tratamentos, sendo quatro cultivares e quatorze genótipos experimentais. Cada parcela teve dez plantas, sendo avaliada a produtividade (t ha-1) e a reação à severidade de danos causados por insetos de solo (por meio de uma escala de notas). Para cada característica foi estimada a estabilidade a adaptabilidade fenotípica pela metodologia proposta por Lin e Binns (1988) e Carneiro (1998). Para produtividade os genótipos BD#52, BD#112 e BD#58 foram identificados como superiores em relação aos demais, sendo os mais adaptados aos ambientes favoráveis para produtividade. Por sua vez, os genótipos BD#22, BD#58 e BD#106 foram mais tolerantes e adaptados a ambientes favoráveis para severidade de insetos de solos. Os genótipos BD#22, BD#58 e BD#106 foram os mais adaptados a ambientes desfavoráveis.

The aim of this study was to estimate the adaptability and stability of sweet potato genotypes evaluated in three environments in the center south state of the Tocantins. The experimental design was a randomized block design with three replications and eighteen treatments, with four cultivars and fourteen experimental clones. Each plot with ten plants was used to evaluate the productivity (t ha-1) and reaction of the incidence of damage caused by soil insects (in a scale of the note). For each trait were estimated stability and phenotypic adaptability by the methodology proposed by Lin e Binns (1988) and Carneiro (1998). For productivity the genotypes BD#52, BD#112 and BD# 58 were identified as adapted to favorable environments of the evaluation. The genotypes BD#22, BD#58 and BD#106 were more tolerant and adapted to favorable environments for the severity of soil insects. Genotypes BD # 22, # 58 and BD BD # 106 were the best adapted to desfavorable environments.