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.

Expression of coffee florigen CaFT1 reveals a sustained floral induction window associated with asynchronous flowering in tropical perennials.

The behavior of florigen(s) and environment-influenced regulatory pathways that control floral initiation in tropical perennials species with complex phenological cycles is poorly understood. Understanding the mechanisms underlying this process is important for food production in the face of climate change, thus, we used Coffea sp. L. (Rubiaceae) as a model to explore this issue. Homologs of FLOWERING LOCUS T (CaFT1) and environment-related regulators CONSTANS (CaCO), PHYTOCHROME INTERACTING FACTOR 4 (CaPIF4) and FLOWERING LOCUS C (CaFLC) were retrieved from coffee genomes and identified through phylogenetic analysis. Overexpression of CaFT1 in Arabidopsis caused early-flowering phenotype and yeast two hybrid studies indicated CaFT1 binding to bZIP floral regulator FD, which suggests that CaFT1 is a coffee florigen. Expression of CaFT1 and other floral regulators, together with carbohydrate analysis, were evaluated over one year using three contrasting genotypes, two C. arabica cultivars and C. canephora. All genotypes showed active and variable CaFT1 transcription from February until October, indicating the potential window for floral induction that reached a maximum in the cold period of June. CaCO expression, as expected, varied over a 24-hour day period and monthly with day length, whereas expression of temperature-responsive homologs, CaFLC and CaPIF4, did not correlate with temperature changes nor CaFT1 expression, suggesting alternative FT regulatory pathways in coffee. Based on our results, we suggest a continuum of floral induction that allows different starting points for floral activation, which explains developmental asynchronicity and prolonged anthesis events in tropical perennial species.

Crosstalk Between Ethylene and Abscisic Acid During Changes in Soil Water Content Reveals a New Role for 1-Aminocyclopropane-1- Carboxylate in Coffee Anthesis Regulation.

Coffee (Coffea arabica L.) presents an asynchronous flowering regulated by an endogenous and environmental stimulus, and anthesis occurs once plants are rehydrated after a period of water deficit. We evaluated the evolution of Abscisic Acid (ABA), ethylene, 1-aminocyclopropane-1-carboxylate (ACC) content, ACC oxidase (ACO) activity, and expression analysis of the Lysine Histidine Transporter 1 (LHT1) transporter, in the roots, leaves, and flower buds from three coffee genotypes (C. arabica L. cv Oeiras, Acauã, and Semperflorens) cultivated under field conditions with two experiments. In a third field experiment, the effect of the exogenous supply of ACC in coffee anthesis was evaluated. We found an increased ACC level, low ACO activity, decreased level of ethylene, and a decreased level of ABA in all tissues from the three coffee genotypes in the re-watering period just before anthesis, and a high expression of the LHT1 in flower buds and leaves. The ethylene content and ACO activity decreased from rainy to dry period whereas the ABA content increased. A higher number of opened and G6 stage flower buds were observed in the treatment with exogenous ACC. The results showed that the interaction of ABA-ACO-ethylene and intercellular ACC transport among the leaves, buds, and roots in coffee favors an increased level of ACC that is most likely, involved as a modulator in coffee anthesis. This study provides evidence that ACC can play an important role independently of ethylene in the anthesis process in a perennial crop.

Elevated Temperatures Impose Transcriptional Constraints and Elicit Intraspecific Differences Between Coffee Genotypes.

The projected impact of global warming on coffee production may require the heat-adapted genotypes in the next decades. To identify cellular strategies in response to warmer temperatures, we compared the effect of elevated temperature on two commercial Coffea arabica L. genotypes exploring leaf physiology, transcriptome, and carbohydrate/protein composition. Growth temperatures were 23/19°C (day/night), as optimal condition (OpT), and 30/26°C (day/night) as a possible warmer scenario (WaT). The cv. Acauã showed lower levels of leaf temperature (Tleaf) under both conditions compared to cv. Catuaí, whereas slightly or no differences for other leaf physiological parameters. Therefore, to explore temperature responsive pathways the leaf transcriptome was examined using RNAseq. Genotypes showed a marked number of differentially-expressed genes (DEGs) under OpT, however DEGs strongly decrease in both at WaT condition indicating a transcriptional constraint. DEGs responsive to WaT revealed shared and genotype-specific genes mostly related to carbohydrate metabolism. Under OpT, leaf starch content was greater in cv. Acauã and, as WaT temperature was imposed, the leaf soluble sugar did not change in contrast to cv. Catuaí, although the levels of leaf starch, sucrose, and leaf protein decreased in both genotypes. These findings revealed intraspecific differences in the underlying transcriptional and metabolic interconnected pathways responsive to warmer temperatures, which is potentially linked to thermotolerance, and thus may be useful as biomarkers in breeding for a changing climate.

Severidade da antracnose e produtividade de sorgo granífero em resposta a doses crescentes de nitrogênio / Anthracnose severity and yield of grain sorghum in response to increasing nitrogen doses

O presente estudo objetivou avaliar a severidade da antracnose e a produtividade de diferentes genótipos de sorgo em resposta a doses crescentes de nitrogênio. Os experimentos foram conduzidos em duas safras agrícolas: safra 2009/2010 (safra I) e safra 2010/2011 (safra II). O preparo do solo na área experimental foi realizado de forma convencional. Na safra I foram utilizados quatro genótipos de sorgo BRS 310, CMSXS 0144015, CMSXS 9920045 e CMSXS 9920044, enquanto que na safra II apenas os genótipos BRS 310 e CMSXS 0144015 foram avaliados. Aos 45 dias após o plantio (DAP) foram aplicados os tratamentos que consistiram de doses de nitrogênio (67; 112; 157; e 202 kg ha-1) em adubação de cobertura. Aos 60 DAP, iniciou-se a avaliação da severidade da antracnose utilizando escala de notas. Na colheita, determinou-se a produtividade de grãos nos tratamentos. Houve variação nos níveis de severidade da antracnose e na produtividade de grãos dos genótipos de sorgo em função das doses de nitrogênio aplicadas. Os genótipos de sorgo CMSXS 9920045 e BRS 310 apresentaram menor e maior suscetibilidade à antracnose, respectivamente. No genótipo BRS 310, a doença progrediu mais rapidamente na safra I que apresentou maior umidade relativa.

The present study aimed to evaluate the severity of anthracnose and yield of different genotypes of sorghum in response to increasing levels of nitrogen. For this, experiments were conducted in two agricultural seasons: crop season 2009/2010 (crop season I) e crop season 2010/2011 (crop season II). The soil preparation in the experimental area was performed in conventional manner. In the crop season I were used four sorghum genotypes BRS 310, CMSXS 0144015, CMSXS 9920045 e CMSXS 9920044, whereas in the crop season II only the genotypes BRS 310 and CMSXS 0144015 were evaluated. At 45 days after planting (DAP) were applied the treatments which consisted of nitrogen doses (67; 112; 157; and 202 kg ha-1) in topdressing. At 60 DAP, was began the evaluation of anthracnose severity using notes scale. At the harvest it was determined the yield for each treatment based on the mass of grain. There was variation in levels of anthracnose severity and grain yield in the sorghum genotypes in response to nitrogen levels applied. The sorghum genotypes CMSXS 9920045 and BRS 310 showed smaller and higher susceptibility to anthracnose, respectively. In genotype BRS 310, the disease progressed more rapidly in the crop season I that showed major relative humidity.

Avaliação in vitro de diferentes métodos de análises de fungitoxicidade de óleos essenciais / Evaluation in vitro of different methods of analysis fungitoxicity of essential oils

Os problemas ambientais causados por fungicidas sintéticos têm elevado as buscas por métodos alternativos de controle de doenças de plantas. Objetivou-se avaliar o efeito do óleo essencial de capim citronela, sobre o fungo Rhizoctonia solani, em diferentes métodos de avaliação de fungitoxicidade in vitro. Foi utilizado delineamento inteiramente casualizado em esquema fatorial com quatro repetições, onde os fatores foram compostos por quatro métodos de avaliação da fungitoxicidade in vitro do óleo essencial (óleo essencial diluído em Tween 80 (0,5%) e incorporado ao meio de cultura BDA (batata, dextrose e ágar) ainda fundente; óleo essencial diluído em Tween 80 (0,5%) e distribuído na superfície do BDA; óleo essencial diluído em Tween 80 (0,5%) e distribuído em papel filtro fixado na superfície interna da tampa da placa de Petri; óleo essencial puro e distribuído na superfície do meio de cultura; e testemunha) e por cinco épocas de avaliação (2, 4, 6, 8 e 10 dias de incubação). Foram utilizados 0,25µL mL-1 do óleo do capim citronela em todos os tratamentos. Dos tratamentos avaliados o uso do óleo puro distribuído na superfície do meio de cultura foi mais eficiente na redução do diâmetro micelial em todas as avaliações. Neste método a taxa de crescimento micelial foi de 9, 02 mm dia-1, atingindo na última época de avaliação 79,77 mm.

Environmental problems caused by synthetic fungicides have increased the search for alternative methods of control of plant diseases. The objective was to evaluate the effect of essential oil of citronella grass, on the fungus Rhizoctonia solani, in different methods of in vitro fungitoxicity. We used a randomized design in a factorial design with four replications, where the factors were composed of four methods for assessing the in vitro fungitoxicity of the essential oil of citronella grass (essential oil diluted in Tween 80 (0.5%) and embedded in the culture medium PDA (potato dextrose agar) still melting, essential oil diluted in Tween 80 (0.5%) and distributed on the surface of the PDA; oil essential diluted in Tween 80 (0.5%) and distributed on filter paper attached to the inner surface of the lid of the Petri dish, pure essential oil and distributed on the surface of the culture medium, and control) and five evaluation periods (2, 4, 6, 8 and 10 days of incubation). Was used 0.25µL mL-1 of citronella oil in all treatments. Of the treatments evaluated the use of pure oil distributed on the surface of the culture medium was more effective in reducing the mycelial diameter in all evaluations. In this method the rate of mycelial growth was 9,02 mm day-1, reaching in last evaluation 79,77 mm.