Plant Tissue Extracts Research Articles

Rapid Detection of the Strawberry Foliar Nematode Aphelenchoides fragariae Using Recombinase Polymerase Amplification Assay with Lateral Flow Dipsticks.

Rapid and reliable diagnostic methods for plant-parasitic nematodes are critical for facilitating the selection of effective control measures. A diagnostic recombinase polymerase amplification (RPA) assay for Aphelenchoides fragariae using a TwistAmp® Basic Kit (TwistDx, Cambridge, UK) and AmplifyRP® Acceler8® Discovery Kit (Agdia, Elkhart, IN, USA) combined with lateral flow dipsticks (LF) has been developed. In this study, a LF-RPA assay was designed that targets the ITS rRNA gene of A. fragariae. This assay enables the specific detection of A. fragariae from crude nematode extracts without a DNA extraction step, and from DNA extracts of plant tissues infected with this nematode species. The LF-RPA assay showed reliable detection within 18-25 min with a sensitivity of 0.03 nematode per reaction tube for crude nematode extracts or 0.3 nematode per reaction tube using plant DNA extracts from 0.1 g of fresh leaves. The LF-RPA assay was developed and validated with a wide range of nematode and plant samples. Aphelenchoides fragariae was identified from seed samples in California. The LF-RPA assay has great potential for nematode diagnostics in the laboratory with minimal available equipment.

Determining Hydrogen Peroxide Content in Plant Tissue Extracts

Determining Hydrogen Peroxide Content in Plant Tissue Extracts

A New Method for Fractionation and Characterization of Polyphenols and Tannins from Grapevine Leaf Tissue.

Plants accumulate different types of phenolic material in their tissue as a response to biotic as well as abiotic stress. Monomeric polyphenols and smaller oligomers can serve as protection against ultraviolet radiation or prevent oxidative tissue damage, while larger molecules such as tannins can be the plant's reaction to an infection or physical damage. Therefore, characterization, profiling, and quantification of diverse phenolics can provide valuable information about the plant and the stress status at any given time. A method was developed that allows the extraction of polyphenols and tannins from leaf tissue, followed by fractionation and quantification. Extraction was performed with liquid nitrogen and 30% acetate-buffered ethanol. The method was tested with four cultivars under varying extraction conditions (solvent strength and temperature) and showed great improvements of the chromatography that would otherwise be impacted by tannins. The separation of tannins from smaller polyphenols was achieved by bovine serum albumin precipitation and resuspension in a urea-triethanolamine buffer. Tannins were reacted with ferric chloride and analyzed spectrophotometrically. Monomeric non-protein-precipitable polyphenols were then analyzed via HPLC-DAD from the supernatant of the precipitation sample. This way, a more complete spectrum of compounds can be analyzed from the same plant tissue extract. With the fractionation suggested here, hydroxycinnamic acids and flavan-3-ols can be separated and quantified with good accuracy and precision. Possible applications include the assessment of plant stress and response monitoring using the total concentrations of polyphenols and tannins, as well as the ratios between those compound classes.

Cis-Prenyltransferases of Marchantia polymorpha: Phylogenetic Analysis and Perspectives for Use as Regulators of Antimicrobial Agent Synthesis

The synthesis of secondary metabolites plays a central role in the survival of plants and their resistance to biotic and abiotic stress. Nevertheless, fundamental and applied studies of plant secondary metabolites, polyisoprenols, remain underdeveloped. The wide distribution of polyisoprenols in plants shows their important role in cellular metabolism. Plant polyisoprenols are synthesized by cis-prenyltransferases (CPTs), the study of which is necessary to understand the synthesis pathways, localization, and functions of plant polyisoprenols. Bryophytes, including the liverwort Marchantia polymorpha, are a unique group of plants with great potential for the study of CPTs. We analyzed the genome of M. polymorpha and identified seven CPT genes, which are homologous to the AtCPT7 and AtCPT3 genes of Arabidopsis thaliana, involved in the synthesis of polyisoprenols. Four individual lines of M. polymorpha plants with mutations in the MpCPT7.37 gene were obtained. It was shown that in three lines the mutation led to a translational frameshift and gene knockout. However, knockout of only the type 7 CPTs had no effect on plant growth and survival. Analysis of the antibacterial activity of mutant plant tissue extracts did not reveal significant changes compared to wild-type tissue extracts, this could be related to a compensatory effect of the activity of other CPTs. These data give us the required base for the further studies of bryophytes CPTs and their products.

Ultra-high performance liquid chromatography Q-Orbitrap MS/MS-based profiling and quantification of limonoids in Meliaceae plants.

Meliaceae plants have been extensively used in agriculture, folklore, and traditional medicine. They are the major storehouses for structurally diverse limonoids (meliacins) possessing various bioactivities like antifeedant, insecticidal, antimicrobial, etc. However accurate detection of these tetranortriterpenes from the vast pool of metabolites in plant tissue extracts or biological sample is a crucial challenge. Though the mass spectrum (MS) provides the molecular mass and the corresponding elemental composition, it cannot be relied precisely. The exact identification of a specific metabolite demands the MS/MS spectrum containing the signature product ions. In the present study, we have developed the UHPLC Q-Orbitrap-based method for identification, quantification, and characterization of limonoids in different plant tissue extracts requiring minimum plant material. Using this method, we carried out the limonoid profiling in different tissue extracts of sixteen Meliaceae plants and the identification of limonoids was performed by comparing the retention time (RT), ESI-( +)-MS spectrum, and HCD-MS/MS of the purified fifteen limonoids used as reference standards. Our results revealed that early intermediates of the limonoid biosynthetic pathway such as azadiradione, epoxyazadiradione, and gedunin occurred more commonly in Meliaceae plants. The MS/MS spectrum library of the fifteen limonoids generated in this study can be utilized for identification of these limonoids in other plant tissue extracts, botanical fertilizers, agrochemical formulations, and bio pesticides.

Tube-Capture (TC) RT-PCR and Multiplex RT-PCR for Diagnosis and Characterization of Viruses in Fruit Trees.

Diagnosis of fruit tree viruses has been challenging for a long time as viral titer is often low and unevenly distributed among different tissues and branches of fruit trees. It is necessary to develop effective and reliable detection systems to identify viral pathogens in fruit trees. In this chapter, I describe RT-PCR and its derivatives tube capture-based reverse-transcription PCR (TC-RT-PCR) and multiplex RT-PCR assays for detection and identification of latent viruses in apple and pear trees. Classical RT-PCR is composed of two steps including transcription of viral RNA using extracted total RNA and PCR amplification of viral cDNA. TC-RT-PCR includes a TC step to capture particles and nucleic acid mixtures from crude plant tissue extracts as template directly for the first single-strand DNA (cDNA) synthesis, followed by PCR to amplify the viral cDNA fragment for viral identification. The cDNA derived from total RNAs can also be used for a one-step multiplex PCR to simultaneously detect several viruses in a given sample. As perennial fruit trees are usually coinfected by several viruses in orchards, multiplex RT-PCR can save time and lower labor and material costs for viral detection. These nucleic acid-based methods are sensitive and may be adapted for detection and identification of diverse viruses from different tissue materials of fruit trees.

Accurate Identification of Bioactive Meliaceae Limonoids by UHPLC-MS/MS Based Structure-Fragment Relationships (SFRs).

Limonoids are bioactive plant specialized metabolites found in the Meliaceae family. The basic limonoids, i.e., azadiradione, epoxyazadiradione, and gedunin have been exploited for various bioactivities and therefore are the potential drug leads for tomorrow. However, their low abundance, structural similarity, and lack of adequate mass fragmentation data have hampered their accurate identification and quantification from various sources. In the present study, basic limonoids such as azadirone, azadiradione, epoxyazadiradione, and gedunin isolated from Neem were utilized for the synthesis of their derivatives and isotopologs. A total of 30 one compounds were used in this study among which five were isolated, two were biotransformed, and 24 were synthesized. Among the synthesized compounds nine are novel compounds including six deuterated analogs/isotopologs which are (1,3-2H)-1,2-dihydro-3β-hydroxyazadiradione (9), (1,3,16-2H)-1,2-dihydro-3β-16β-dihydroxyazadiradione (10), 3β-hydroxyazadiradione (11), 3β-16β-dihydroxyazadiradione (12), (3-2H)-3β-hydroxyazadiradione (13), (3,16-2H)-3β-16β-dihydroxyazadiradione (14), (1,3,7-2H)-1,2-dihydro-3β-hydroxy-7-deacetylazadiradione (15), 1,2,20,21,22,23-hexahydroazadiradione (17), and (1,3-2H)-1,2-dihydro-3β-hydroxygedunin (29). These limonoids along with their semisynthesized derivatives were subjected to ultra high performance liquid chromatography mass spectrometry (UHPLC–MS/MS) and the fragmentation pathway was established based on structure–fragment relationships (SFRs), utilizing high resolution MS/MS data. We have developed a most reliable and easily reproducible protocol describing in depth analysis of SFRs based on the structural modifications and synthesis of isotopologs. Also, the MS/MS fragment library of these basic limonoids generated in this study acts as a fingerprint for accurate identification and quantification of limonoids by MS/MS analysis in various plant tissue extracts, phytopharmaceutical formulations and biological samples.

METHOD FOR DETERMINING THE TOTAL CONTENT OF PHENOLIC COMPOUNDS IN PLANT EXTRACTS WITH FOLIN-DENIS REAGENT AND FOLIN-CHOCALTEU REAGENT: MODIFICATION AND COMPARISON

A modification of the method for determining the total content of phenolic compounds in plant tissue extracts with the Folin-Denis reagent and the Folin-Ciocalteu reagent has been carried out, allowing to establish the correspondence of the results obtained when using them. The method using the Folin-Denis reagent is adapted for conducting determinations in microvolumes. For the method using the Folin-Ciocalteu reagent, the concentration of the latter (0.4 N, a 5-fold dilution of the standard reagent) and the composition of the reaction mixture were selected, using which the optical densities of the reduction products of the Folin-Denis and Folin-Ciocalteu reagents containing polyphenols in ethanol extracts from wheat, buckwheat and calus tissue of tea were almost the same. The absorption spectra of the reduction products of these reagents by gallic acid, rutin, (-)-epicatechin, as well as ethanol extracts from wheat, buckwheat, and tea calus tissue, were located in the same region (680–770 nm) and had similar characteristics. Calibration graphs of the dependence of the optical density of solutions on the concentration of standard substances (gallic acid, (-)-epicatechin, rutin), constructed using the Folin-Denis and Folin-Ciocalteu reagents, had a linear character within the concentration range of 10–100 μg/ml and practically coincided. The results of determining the content of phenolic compounds in ethanol extracts of plants, differing in their ability to accumulate, showed very similar and statistically significant values when using these two reagents.

Mid-infrared spectroscopy as process analytical technology tool for estimation of THC and CBD content in Cannabis flowers and extracts

Tetrahydrocannabinol (THC) and cannabidiol (CBD) are the most notable Cannabis components with pharmacological activity and their content in the plant flowers and extracts are considered as critical quality parameters. The new Medical Cannabis industry needs to adopt the quality standards of the pharmaceutical industry, however, the variability of phytocannabinoids content in the plant material often exerts an issue in the inconsistency of the finished product quality parameters. Sampling problems and sample representativeness is a major limitation in the end-point testing, particularly when the expected variation of the product quality parameters is high. Therefore, there is an obvious need for the introduction of Process Analytical Technology (PAT) for continuous monitoring of the critical quality parameters throughout the production processes. Infrared spectroscopy is a promising analytical technique that is consistent with the PAT requirements and its implementation depends on the advances in instrumentation and chemometrics that will facilitate the qualitative and quantitative aspects of the technique. Our present work aims in highlighting the potential of mid-infrared (MIR) spectroscopy as PAT in the quantification of the main phytocannabinoids (THC and CBD), considered as critical quality/material parameters in the production of Cannabis plant and extract. A detailed assignment of the bands related to the molecules of interest (THC, CBD) was performed, the spectral features of the decarboxylation of native flowers were identified, and the specified bands for the acid forms (THCA, CBDA) were assigned and thoroughly explained. Further, multivariate models were constructed for the prediction of both THC and CBD content in extract and flower samples from various origins, and their prediction ability was tested on a separate sample set. Savitskzy-Golay smoothing and the second derivative of the native MIR spectra (1800–400 cm−1 region) resulted in best-fit parameters. The PLS models presented satisfactory R2Y and RMSEP of 0.95 and 3.79% for THC, 0.99 and 1.44% for CBD in the Cannabis extract samples, respectively. Similar statistical indicators were noted for the Partial least-squares (PLS) models for THC and CBD prediction of decarboxylated Cannabis flowers (R2Y and RMSEP were 0.99 and 2.32% for THC, 0.99 and 1.33% for CBD respectively). The VIP plots of all models demonstrated that the THC and CBD distinctive band regions bared the highest importance for predicting the content of the molecules of interest in the respected PLS models. The complexity of the sample (plant tissue or plant extract), the variability of the samples regarding their origin and horticultural maturity, as well as the non-uniformity of the plant material and the flower-ATR crystal contact (in the case of Cannabis flowers) were governing the accuracy descriptors. Taking into account the presented results, ATR-MIR should be considered as a promising PAT tool for THC and CBD content estimation, in terms of critical material and quality parameters for Cannabis flowers and extracts.

Padlock probe-based rolling circle amplification lateral flow assay for point-of-need nucleic acid detection.

Sensitive, reliable and cost-effective detection of pathogens has wide ranging applications in clinical diagnostics and therapeutics, water and food safety, environmental monitoring, biosafety and epidemiology. Nucleic acid amplification tests (NAATs) such as PCR and isothermal amplification methods provide excellent analytical performance and significantly faster turnaround times than conventional culture-based methods. However, the inherent cost and complexity of NAATs limit their application in resource-limited settings and the developing world. To help address this urgent need, we have developed a sensitive method for nucleic acid analysis based on padlock probe rolling circle amplification (PLRCA), nuclease protection (NP) and lateral flow detection (LFA), referred to as PLAN-LFA, that can be used in resource-limited settings. The assay involves solution-phase hybridization of a padlock probe to target, sequence-specific ligation of the probe to form a circular template that undergoes isothermal rolling circle amplification in the presence of a polymerase and a labeled probe DNA. The RCA product is a long, linear concatenated single-stranded DNA that contains binding sites for the labeled probe. The sample is then exposed to a nuclease which selectively cleaves single-stranded DNA, the double-stranded labeled probe is protected from nuclease digestion and detected in a lateral flow immunoassay format to provide a visual, colorimetric readout of results. We have developed specific assays targeting beta-lactamase resistance gene for monitoring of antimicrobial resistance and Severe Acute Respiratory Syndrome-Coronavirus-2 (SARS-CoV-2, the novel coronavirus discovered in 2019) using the PLAN-LFA platform. The assay provides a limit of detection of 1.1 pM target DNA (or 1.3 × 106 copies per reaction). We also demonstrate the versatility and robustness of the method by performing analysis on DNA and RNA targets, and perform analysis in complex sample matrices like saliva, plant tissue extract and bacterial culture without any sample pretreatment steps.

Development and validation of a UHPLC-ESI-QTOF mass spectrometry method to analyze opines, plant biomarkers of crown gall or hairy root diseases

Opines are low-molecular-weight metabolites specifically biosynthesized by agrobacteria-transformed plant cells when plants are struck by crown gall and hairy root diseases, which cause uncontrolled tissue overgrowth. Transferred DNA is sustainably incorporated into the genomes of the transformed plant cells, so that opines constitute a persistent biomarker of plant infection by pathogenic agrobacteria and can be targeted for crown gall/hairy root disease diagnosis. We developed a general, rapid, specific and sensitive analytical method for overall opine detection using ultra-high-performance liquid chromatography-electrospray ionization quadrupole time-of-flight mass spectrometry (UHPLC-ESI-MS-QTOF), with easy preparation of samples. Based on MS, MS/MS and chromatography data, the detection selectivity of a wide range of standard opines was validated in pure solution and in different plant extracts. The method was successfully used to detect different structural types of opines, including opines for which standard compounds are unavailable, in tumors or hairy roots induced by pathogenic strains. As the method can detect a wide range of opines in a single run, it represents a powerful tool for plant gall analysis and crown gall/hairy root disease diagnosis. Using an appropriate dilution of plant extract and a matrix-based calibration curve, the quantification ability of the method was validated for three opines belonging to different families (nopaline, octopine, mannopine), which were accurately quantified in plant tissue extracts.

Influence of thermal modification and extraction techniques on yield, antioxidant capacity and phytochemical profile of chestnut (Castanea sativa Mill.) wood

Abstract Numerous investigations on the antioxidant properties of different plant tissue extracts are available in literature, but few studies on the effect of thermally modified wood on secondary metabolites were carried out up to now. This study highlights the influence of the thermal modification of Castanea sativa Mill. wood on total content of antioxidant compounds and consequently on its antioxidant activity. In addition, a chemical profile by Gas Chromatography–Mass Spectrometry (GC–MS) of the extractives was carried out. Wood of chestnut, thermally modified at 180 °C for 3 h, was used to obtain wood meal which was subjected to different extraction techniques, as maceration extraction (ME), ultrasound assisted extraction (UAE) and accelerated solvent extraction (ASE). The total content of principal antioxidant compounds, such as polyphenols, flavonoids and tannins, as well as the evaluation of antioxidant capacity by using different in vitro assays were determined. Relative Antioxidant Capacity Index (RACI), which is used to compare all antioxidant parameters, has also been applied. The study demonstrated a positive influence on chemical compounds present in C. sativa Mill wood originating from the thermal modification process. Thus, is possible to consider thermal modification as a promising strategy to improve the antioxidant activity of chestnut wood extractives.

Iron-Chelating Potential of Novel Phytochemicals in Poplar and Cedar Trees

Introduction: Iron overload is not only a consequence of diseases such as thalassemia and hereditary hemochromatosis (HFE), but also of neurodegeneration with brain iron accumulation (NBIA). In HFE, iron increases the risk of developing hepatocellular and colorectal cancers. Excess iron resulting from disruptions in normal iron homeostasis can accumulate in major organs including liver, heart and brain, and has devastating effects if left untreated. Currently, treatment includes using iron chelators, which at higher concentrations can have significant adverse effects and require constant medical supervision. Therefore, the search for alternative unique or adjuvant iron chelators that have reduced toxicity could be of significant benefit. Plants that grow in alkaline soils may be a good source of chelators for this purpose. Since iron is generally unavailable in such soils, plant roots have evolved mechanisms to solubilize iron for uptake, such as soil acidification, but need additional strategies to overcome high alkalinity. This may include producing secondary metabolites that are exuded into the soil and can chelate iron directly, including phenolic acids that may chelate iron at physiological pH in humans. This project is focused on finding, isolating, and testing bioactivity of compounds from western red cedar (alkaline tolerant) and poplar (rich in phenolics). Methods: Plants are grown in iron-normal and -deficient conditions in an innovative aeroponic system to stimulate the production of secondary metabolites related to Fe deficiency. Plant tissue extracts and root washings are collected and concentrated with solid phase extraction chromatography to form plant-derived concentrates (PDC) that are analyzed by UPLC-MS and colourimetric assays to isolate, identify, and characterize compounds induced by iron-deficiency. For bio-activity testing, PDCs are introduced to cultures of THP-1 cells, a model human monocytic cell line, to study their effect on Fe homeostasis. Prior to treatment with chelators, cells are cultured under normal (Con) and Fe-overload (CrFe) conditions (produced by treatment with 10 and 20 µM Fe-citrate) for one week to model human chronic iron overload. Deferoxamine (DFO), a well-known clinical iron chelator, model phenolics like caffeic (CafA) and chlorogenic acid (CGA), and PDCs have been applied to cultures as potential chelators. Results: Leaf compared to root tissues from poplar vary greatly in their CGA and phenolic content. Leaf extracts contained 5 times more phenolics than root extracts, and root extracts from iron-deficient plants produced 66% more phenolic compounds than those from iron-normal plants. Compared to leaf extracts, root extracts showed a 4-fold increase in iron-binding activity in vitro. PDCs including these extracts were found to contain compounds responsive to iron deficiency, which are semi-polar and low in molecular weight (140 - 340 m/z). Distinct iron-responsive compounds were also identified from cedar. Following acute dosage with Fe-citrate, THP-1 cells showed a moderate reduction in iron content after treatment with CafA, CGA, and PDCs from roots, with no influence on cell viability. On-going work includes dose-dependency with CafA and PDCs and co-operative effects of PDCs with DFO. Iron-loading in THP-1 cells is time sensitive, with maximum iron uptake measured at 8 hours following delivery of 20 µM Fe-citrate. Detailed kinetics of cellular iron-loading in the presence of iron-chelators is currently being investigated. Conclusions: We found that low-molecular weight and water-soluble PDCs from iron-deficient plants had excellent iron-binding capacity in vitro, and inhibited iron uptake in THP-1 cells. Effects of chelators on cellular iron uptake is both dose and time dependent. Screening plants for novel chelators provides an abundance of opportunity to search for new chelators for human medicinal use. Disclosures No relevant conflicts of interest to declare.

Detection of a secreted protein biomarker for citrus Huanglongbing using a single-walled carbon nanotubes-based chemiresistive biosensor

Citrus greening, or Huanglongbing (HLB), is currently the most devasting disease of citrus, creating unprecedented crisis for the multibillion-dollar global citrus industry. To-date, there is no effective cure and disease management relies on early detection and removal of infected trees. Thus, it is imperative that accurate, timely, and robust disease detection and diagnosis technologies are available to minimize the spread of disease. This study reports a sensitive and selective label-free biosensor that combines the physical and chemical advantages of carbon nanomaterials like single-walled carbon nanotubes (SWNTs) in a field-effect transistor (FET)/chemiresistor architecture with selective antibodies against Sec-delivered effector 1 (SDE1), a secreted protein biomarker, for the detection of HLB. The biosensor detected SDE1 biomarkers for citrus greening in plant tissue extracts with the dynamic range over three orders of magnitude in the low nanomolar to micromolar concentration range and limit of detection of 5 nM. The study also demonstrated the use of the standard additions assay method with the biosensor to attain a 90-percent signal recovery in concentrated plant tissue extract, allowing for quantitative detection without an external calibration. Adopting the novel detection strategy targeting the secreted protein biomarker, SDE1, addresses some of the challenges faced by current methods of nucleic acid-based assays and symptom-based diagnosis, which have been found prone to false negatives and misdiagnoses, respectively.

Drying Enhances Signal Intensities for Global GC⁻MS Metabolomics.

We report here that a straightforward change of the standard derivatization procedure for GC–MS metabolomics is leading to a strong increase in metabolite signal intensity. Drying samples between methoxymation and trimethylsilylation significantly increased signals by two- to tenfold in extracts of yeast cells, plant and animal tissue, and human urine. This easy step reduces the cost of sample material and the need for expensive new hardware.

Inverse Molecular Sentinel-Integrated Fiberoptic Sensor for Direct and in Situ Detection of miRNA Targets.

Molecular advances have been made in analysis systems for a wide variety of applications ranging from biodiagnostics, biosafety, bioengineering, and biofuel research applications. There are, however, limited practical tools necessary for in situ and accurate detection of nucleic acid targets during field work. New technology is needed to translate these molecular advances from laboratory settings into the real-life practical monitoring realm. The exquisite characteristics (e.g., sensitivity and adaptability) of plasmonic nanosensors have made them attractive candidates for field-ready sensing applications. Herein, we have developed a fiber-based plasmonic sensor capable of direct detection (i.e., no washing steps required) of nucleic acid targets, which can be detected simply by immerging the sensor in the sample solution. This sensor is composed of an optical fiber that is decorated with plasmonic nanoprobes based on silver-coated gold nanostars (AuNS@Ag) to detect target nucleic acids using the surface-enhanced Raman scattering (SERS) sensing mechanism of nanoprobes referred to as inverse molecular sentinels (iMS). These fiber-optrodes can be reused for several detection-regeneration cycles (>6). The usefulness and applicability of the iMS fiber-sensors was tested by detecting target miRNA in extracts from leaves of plants that were induced to have different expression levels of miRNA targets. These fiber-optrodes enable direct detection of miRNA in plant tissue extract without the need for complex assays by simply immersing the fiber in the sample solution. The results indicate the fiber-based sensors developed herein have the potential to be a powerful tool for field and in situ analysis of nucleic acid samples.

Detection of ‘Candidatus Liberibacter asiaticus’ in citrus by concurrent tissue print‐based qPCR and immunoassay

‘Candidatus Liberibacter asiaticus’ (CLas) is associated with the most destructive disease of citrus, huanglongbing (HLB). The most widely used methods for detection of CLas are PCR‐based and require purification of DNA from plant samples. Elution of DNA from tissue prints made on nitrocellulose membranes followed by qPCR (TPE‐qPCR) was compared to DNA extraction of plant tissue followed by PCR (X‐PCR) by testing the same tissue samples. The former estimated a higher CLas population in tissue prints than the latter (t‐test; P = 0.009). All extracts prepared for TPE‐qPCR throughout the experiment were also tested by conventional PCR and 80.8% were identified as positive. A similar set of stem and petiole tissue samples was tested by TPE‐qPCR and immunoassay. Although the detection rate by TPE‐qPCR was higher than by immunoassay, about 6% of tissue prints were positive by immunoassay but not by TPE‐qPCR. Thus, a higher detection rate would be achieved by combining TPE‐qPCR with immunoassay. Significant differences were observed in the performance of nitrocellulose membranes from different manufacturers in these assays. Immunotissue prints showed that the spatial distribution pattern of CLas infection varied widely from one sample to another, but the patterns were highly correlated among serial sections from the same sample, suggesting that CLas preferentially colonizes adjacent phloem cells in a vertical rather than horizontal direction.

Pyrrolizidine Alkaloids-Free Extract from the Cell Culture of Lithospermum officinale with High Antioxidant Capacity.

The benefits of Lithospermum officinale has encouraged people to continue using its extract (CAS 90063-58-4) in both medicinal and cosmetic industries despite the fact that chemical analysis confirms the presence of pyrrolizidine alkaloids (PAs) in the extract. While the cultivation of L. officinale takes, at least, 2years to produce usable crops, its callus culture proliferated 8.3 times with 4.9-fold biomass in less than 30days under the applied conditions in this study. Under the applied conditions, the cell extract contained no toxic PAs while phenylpropanoid pathway was active toward phenolic acids formation not toward naphthoquinone derivatives. Rosmarinic acid was produced as the main constituent. Total phenolic content and antioxidant capacity of the proliferated cell extracts were similar to those of the extracts of the natural plant tissues, in particular from the root. These results support the idea that the extract of L. officinale cells can be a reliable substitute for the extract of the natural plant tissues.

Effects of Psidium guajava leaf extract on secretion systems of Gram-negative enteropathogenic bacteria.

We screened a total of 672 plant-tissue extracts to search for phytochemicals that inhibit the function of the type III secretion system (T3SS) of enteropathogenic Escherichia coli (EPEC) and enterohemorrhagic E. coli (EHEC). Among candidates examined, we found that an extract from the leaves of Psidium guajava (guava) inhibited the secretion of the EspB protein from EPEC and EHEC without affecting bacterial growth. The guava extract (GE) also inhibited EPEC and EHEC from adhering to and injecting EspB protein into HEp-2 cells. GE seemed to block the translocation of EspB from the bacterial cells to the culture medium. In addition to EPEC and EHEC, GE also inhibited the T3SS of Yersinia pseudotuberculosis and Salmonella enterica serovar Typhimurium. After exposure to GE, Y. pseudotuberculosis stopped the secretion of Yop proteins and lost its ability to induce the apoptosis of mouse bone marrow-derived macrophages. S. Typhimurium exposed to GE ceased the secretion of Sip proteins and lost its ability to invade HEp-2 cells. GE inhibited EspC secretion, the type V secretion protein of EPEC, but not Shiga toxin2 from EHEC. Thus, our results suggest that guava leaves contain a novel type of antimicrobial compound that could be used for the therapeutic treatment and prevention of gram-negative enteropathogenic bacterial infections.

A thaumatin-like genomic sequence identification in Vitis vinifera l., stormy wines and musts based on direct pcr

Direct polymerase chain reaction method was use to amplify a thaumatin-like sequence of Vitis vinifera L. in grapes as well as in stormy wines and musts. Thaumatin-like proteins (TLPs) of Vitis vinifera possess beside its function in abiotic and biotic stress response another one - they are able to cause protein haze in wine unless removed prior to bottling. Direct PCR is an approach where omission of DNA extraction is typical prior the amplification of the target site of plant genome. Crude extract or small pieces of plant tissues are used in the analysis directly without steps of extraction and purification of gDNA. The biological material that was used in analysis was collected during August - October 2017 in local stores and winery Sabo and comprises from cultivars Iršai, Muškát, Savignon Blanc, Svätovavrinecké, Dornfelder and Pálava. Direct PCR was performed by a cutted piece of grape tissue and a dilution buffer was use in 1:2 for stormy wine or must, respectively. Direct amplification of thaumatin-like protein sequence of Vitis vinifera was performed along with the control reactions with the primers for conserved region of plant chloroplast. Possitive amplification of thaumatin-like allergen sequence resulted in 570 bp amplicon. The most abundant amplicons were amplified in stormy wines, followed by musts and the amplicons from grapes were weaker when comparing them to others. The amplicon specificity checking of obtained PCR product of thaumatin-like allergen was performed by restriction cleavage by Psi I and resulted in restriction amplicons of the 80 bp, 81 bp, 94 bp and 315 bp in length. Confirmation of the amplicon specificity by restriction cleavage support the potential of direct PCR to become a reproducible method that will be fully applicable in routine analysis of not only plant genomes in the future, but it was demonstrated, that it works in liquids, too.