ReDivia - Repositorio Digital de l'Instit Valencià d'Investigacions Agràries
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A Novel and Highly Inclusive Quantitative Real-Time RT-PCR Method for the Broad and Efficient Detection of Grapevine Leafroll Associated Virus 1
Get PDFGrapevine (Vitis vinifera L.) is one of the most important crops in the world due to its economic and social impact. Like many other crops, grapevine is susceptible to different types of diseases caused by pathogenic microorganisms. Grapevine leafroll-associated virus 1 (GLRaV-1) is a virus associated with grapevine leafroll disease and it is considered at the national and European level as a pathogen that must be absent in propagative plant material. For this reason, the availability of specific, sensitive and reliable detection techniques to ascertain the sanitary status of the plants is of great importance. The objective of this research was the development of a new GLRaV-1 detection method based on a TaqMan quantitative real-time RT-PCR targeted to the coat protein genomic region and including a host internal control in a duplex reaction. To this end, three new GLRaV-1 full genomes were recovered by HTS and aligned with all sequences available in the databases. The method has been validated following EPPO standards and applied for the diagnosis of field plant material and transmission vectors. The new protocol designed has turned out to be highly sensitive as well as much more specific than the current available methods for the detection and absolute quantitation of GLRaV-1 viral titer
Zoning strategies for managing outbreaks of alien plant pests in the European Union: a review
Get PDFManaging outbreaks of alien plant pests is key to preserving biodiversity and safeguarding crop production. Zoning strategies are applied by plant health authorities to tailor measures to the risk of spread in relation to distance from the outbreak epicentre and the biology of the pest. Here we synthesize information on outbreak management to evaluate the diversity and consistency of such approaches. We collected information on the zoning strategies of 121 outbreaks of 25 plant pests in the European Union (EU). According to the organism’s presence and the measures applied, five zones were distinguished: an infested zone (83% of cases), a buffer zone (76%), a clear-cutting zone (28%), an eradication zone (1%) and a containment zone (1%). Infested zones and buffer zones were adjacent non-overlapping zones, while the clear-cutting zone, eradication zone or containment zone was within either the infested zone or buffer zone. A combination of infested and buffer zones was used in 51% of recorded cases. Measures differed within different zones. Destruction of infested plants in the infested zone was done in 78% of the cases, while surveillance was always applied in the buffer zone. Regulation of an organism at EU level led to a convergence of zoning strategies applied by different member states. Regulations often prescribed the
greatest widths used before regulations were issued. Further analyses are needed to explore the efficacy of different strategies including the costs of each strategy. Such analyses should combine insight from practice with bio-economic modelling
Development and Validation of a Duplex RT-qPCR for Detection of Peach Latent Mosaic Viroid and Comparison of Different Nucleic-Acid-Extraction Protocols
Get PDFPeach latent mosaic viroid (PLMVd) is an important pathogen that causes disease in peaches. Control of this viroid remains problematic because most PLMVd variants are symptomless, and although there are many detection tests in use, the reliability of PCR-based methods is compromised by the complex, branched secondary RNA structure of the viroid and its genetic diversity. In this study, a duplex RT-qPCR method was developed and validated against two previously published single RT-qPCRs, which were potentially able to detect all known PLMVd variants when used in tandem. In addition, in order to simplify the sample preparation, rapid-extraction protocols based on the use of crude sap or tissue printing were compared with commercially available RNA purification kits. The performance of the new procedure was evaluated in a test performance study involving five participant laboratories. The new method, in combination with rapid-sample-preparation approaches, was demonstrated to be feasible and reliable, with the advantage of detecting all different PLMVd isolates/variants assayed in a single reaction, reducing costs for routine diagnosis
Estimation of Macro and Micronutrients in Persimmon (Diospyros kaki L.) cv. ‘Rojo Brillante’ Leaves through Vis-NIR Reflectance Spectroscopy
Get PDFThe nutritional diagnosis of crops is carried out through costly elemental analyses of different plant organs, particularly leaves, in the laboratory. However, visible and near-infrared (Vis-NIR) spectroscopy of unprocessed plant samples has a high potential as a faster, non-destructive, environmental-friendly alternative to elemental analyses. In this work, the potential of this technique to estimate the concentrations of macronutrients such as nitrogen (N), phosphorus (P), potassium (K), calcium (Ca), and magnesium (Mg), and micronutrients such as iron (Fe), manganese (Mn) and boron (B), in persimmon (Diospyros kaki L.) ‘Rojo Brillante’ leaves, has been investigated. Throughout the crop cycle variable rates of N and K were applied to obtain six nutritional status levels in persimmon trees in an experimental orchard. Then, leaves were systematically sampled throughout the cropping season from the different nutritional levels and spectral reflectance measurements were acquired in the 430–1040 nm wavelength range. The concentrations of nutrients were determined by inductively coupled plasma optical emission spectrometry (ICP-OES) for P, K, Ca, Mg, Fe, Mn and B after microwave digestion, while the Kjeldahl method was used for N. Then, partial least squares regression (PLS-R) was used to model the concentrations of these nutrients from the reflectance measurements of the leaves. The model was calibrated using 75% of the samples while the remaining 25% were left as the independent test set for external validation. The results of the test set indicated an acceptable validation for most of the nutrients, with determination coefficients (R2) of 0.74 for N and P, 0.54 for K, 0.77 for Ca, 0.60 for Mg, 0.39 for Fe, 0.69 for Mn and 0.83 for B. These findings support the potential use of Vis-NIR spectrometric techniques as an alternative to conventional laboratory methods for the persimmon nutritional status diagnosis although more research is needed to know how the models developed one year perform in ensuing years
Qualitative, energy and environmental aspects of microwave drying of pre-treated apple slices
Get PDFIn the present research, response parameters such as specific energy consumption (SEC), thermal efficiency (TE), energy efficiency (EF), drying time (DT), greenhouse gas (GHG) emission (such as CO2 and NOx), and quality features (color variation and shrinkage) were modeled by response surface methodology (RSM) for apple slices dried in a microwave dryer under ultrasonication (30 ℃-10 min) and blanching (80 °C-2 min) pretreatments. Also, RSM was applied to optimize two independent parameters including microwave power and sample thickness in the levels 100, 200, and 300 W and 2, 4, and 6 mm, respectively. The results indicated the significant influence (P < 0.01) of the independent parameters on the response parameters. The vales of SEC, DT, GHG emission, shrinkage, and color difference were linearly decreased with the declining sample thickness and increasing microwave power, while the energy and thermal efficiencies were increased by a quadratic equation. The use of ultrasonication and blanching pretreatments decreased the SEC, GHG emissions, and DT; while improving the quality of the samples as compared to the non-treated slices. The optimization results showed the optimal drying times (31.55, 82.19, and 50.55 min), SEC (3.42, 10.07, and 4.37 MJ/kg), CO2 with natural gas (1539.75, 1518.75, and 4585 g), CO2 with gas oil (3662.53, 2099.25, 2721.25 g), NOx with natural gas (10.094, 9.956, and 12.906 g), and NOx with gas oil (12.934, 12.758, and 16.538 g) at a microwave power of 300 W and sample thickness of 2 mm with desirability of 0.921, 0.935, and 0.916 for control samples, ultrasonicated, and blanched, respectively
Evaluation of hot water and GRAS salt solutions for the control of postharvest gray and green molds of pomegranate fruit
Get PDFGray and green molds, caused by the fungi Botrytis cinerea and Penicillium spp., respectively, are among the most important postharvest diseases of pomegranate fruit in Spain. Although the synthetic fungicide fludioxonil is allowed for postharvest use since 2019, disease control methods alternative to agrochemicals are preferred, especially in pomegranate export markets, due to health and environmental issues associated with chemical residues. In this work, the disease control ability of dips in hot water (20, 40, 50, 55, 60, 65, 70, and 75 ºC) or aqueous solutions of GRAS (generally recognized as safe) salts (PS, potassium sorbate; SC, sodium carbonate; SBC, sodium bicarbonate; SB, sodium benzoate; all at 3% (w/v)) was evaluated using ‘Mollar de Elche’ pomegranates artificially inoculated with the pathogens B. cinerea or P. sclerotiorum
Rapid Prediction of Nutrient Concentration in Citrus Leaves Using Vis-NIR Spectroscopy
Get PDFThe nutritional diagnosis of crops is carried out through costly foliar ionomic analysis in laboratories. However, spectroscopy is a sensing technique that could replace these destructive analyses for monitoring nutritional status. This work aimed to develop a calibration model to predict the foliar concentrations of macro and micronutrients in citrus plantations based on rapid non-destructive spectral measurements. To this end, 592 ‘Clementina de Nules’ citrus leaves were collected during several months of growth. In these foliar samples, the spectral absorbance (430–1040 nm) was measured using a portable spectrometer, and the foliar ionomics was determined by emission spectrometry (ICP-OES) for macro and micronutrients, and the Kjeldahl method to quantify N. Models based on partial least squares regression (PLS-R) were calibrated to predict the content of macro and micronutrients in the leaves. The determination coefficients obtained in the model test were between 0.31 and 0.69, the highest values being found for P, K, and B (0.60, 0.63, and 0.69, respectively). Furthermore, the important P, K, and B wavelengths were evaluated using the weighted regression coefficients (BW) obtained from the PLS-R model. The results showed that the selected wavelengths were all in the visible region (430–750 nm) related to foliage pigments. The results indicate that this technique is promising for rapid and non-destructive foliar macro and micronutrient prediction
RNAi-mediated silencing of Mediterranean fruit fly (Ceratitis capitata) endogenous genes using orally-supplied double-stranded RNAs produced in Escherichia coli
Get PDFBACKGROUND: The Mediterranean fruit fly (medfly), Ceratitis capitata Wiedemann, is a major pest affecting fruit and vegetable production worldwide, whose control is mainly based on insecticides. Double-stranded RNA (dsRNA) able to down-regulate endogenous genes, thus affecting essential vital functions via RNA interference (RNAi) in pests and pathogens, is envisioned as a more specific and environmentally-friendly alternative to traditional insecticides. However, this strategy has not been explored in medfly yet. RESULTS: Here, we screened seven candidate target genes by injecting in adult medflies gene-specific dsRNA hairpins transcribed in vitro. Several genes were significantly down-regulated, resulting in increased insect mortality compared to flies treated with a control dsRNA targeting the green fluorescent protein (GFP) complementary DNA (cDNA). Three of the dsRNAs, homologous to the beta subunit of adenosine triphosphate (ATP) synthase (ATPsynbeta), a vacuolar ATPase (V-ATPase), and the ribosomal protein S13 (RPS13), were able to halve the probability of survival in only 48 h after injection. We then produced new versions of these three dsRNAs and that of the GFP control as circular molecules in Escherichia coli using a two-self-splicing-intron-based expression system and tested them as orally-delivered insecticidal compounds against medfly adults. We observed a significant down-regulation of V-ATPase and RPS13 messenger RNAs (mRNAs) (approximately 30% and 90%, respectively) compared with the control medflies after 3 days of treatment. No significant mortality was recorded in medflies, but egg laying and hatching reduction was achieved by silencing V-ATPase and RPS13. CONCLUSION: In sum, we report the potential of dsRNA molecules as oral insecticide in medfly
Urban Honey: A Review of Its Physical, Chemical, and Biological Parameters That Connect It to the Environment
Get PDFHumans mainly use the land for agriculture and housing, resulting in the loss of natural habitat and a decrease in the number of species, including wild bees. The reduction of wild bees generates several negative consequences for the agricultural and ecosystem contexts, although sometimes the farming reduces the probability of abandonment of the land. In parallel, urban beekeeping has emerged and consolidated as one of the current trends, while the consumption of honey from urban environments is also growing. Current scientific literature establishes different physical, chemical, and biological parameters which evaluate the quality of honey products and its environment. The review carried out here collects the various compounds contained in this source-dependent food matrix from anthropogenic activities in the sampling area. Using gas and liquid chromatography and spectrometry, the main physicochemical parameters have been detected, 27 chemical elements, of which 6 are heavy elements, 16 polycyclic aromatic hydrocarbons (PAHs), polychlorinated biphenyls (PCBs), and pesticides (organochlorine pesticides and neonicotinoids). Additionally, their total phenolic and microbiological content has been typified. This analysis can help to frame the main characteristics to evaluate this universal product, whose consumption began with the first settlers of the world, and its properties have been evolving as well as the characteristics of the production systems
Short-term water stress responses of grafted pepper plants are associated with changes in the hormonal balance
Get PDFPhytohormones play an important role in regulating the plant behavior to drought. In previous studies, NIBER® pepper rootstock showed tolerance to drought in terms of production and fruit quality compared to ungrafted plants. In this study, our hypothesis was that short-term exposure to water stress in young, grafted pepper plants would shed light on tolerance to drought in terms of modulation of the hormonal balance. To validate this hypothesis, fresh weight, water use efficiency (WUE) and the main hormone classes were analyzed in self-grafted pepper plants (variety onto variety, V/V) and variety grafted onto NIBER® (V/N) at 4, 24, and 48h after severe water stress was induced by PEG addition. After 48h, WUE in V/N was higher than in V/V, due to major stomata closure to maintain water retention in the leaves. This can be explained by the higher abscisic acid (ABA) levels observed in the leaves of V/N plants. Despite the interaction between ABA and the ethylene precursor, 1-aminocyclopropane-1-carboxylic acid (ACC), in relation to stomata closure is controversial, we observed an important increase of ACC at the end of the experiment in V/N plants coinciding with an important rise of the WUE and ABA. The maximum concentration of jasmonic acid and salicylic acid after 48h was found in the leaves of V/N, associated with their role in abiotic stress signaling and tolerance. Respect to auxins and cytokinins, the highest concentrations were linked to water stress and NIBER®, but this effect did not occur for gibberellins. These results show that hormone balance was affected by water stress and rootstock genotype, where NIBER® rootstock displayed a better ability to overcome short-term water stress