Identification and Genome Characterization of a Dahlia Common Mosaic Virus Isolate from China.

Dahlia (Dahlia variabilis) is a widely cultivated ornamental and medicinal plant in China. Recently, dahlia plants with symptoms of leaf mottling and distortion were collected in Hohhot, Inner Mongolia, China. The presence of dahlia common mosaic virus (DCMV), an unassigned species in the genus Caulimovirus, was confirmed by high-throughput sequencing. Three fragments of DCMV Inner Mongolia isolate (DCMV-IN) were PCR-amplified with specific primers, sequenced and assembled into the complete genome sequence with a GenBank accession number of OR494328. The double-stranded circular DNA genome of DCMV-IN consists of 7949 bp and contains six open reading frames (ORFs). Sequence analysis showed that DCMV-IN shared high sequence identities with other DCMV isolates available in the GenBank database. Phylogenetic analysis of DCMV isolates and other representative caulimoviruses based on genome sequence clustered four DCMV isolates to a single branch which was closest to dahlia mosaic virus (DMV). No recombination event was detected among the four DCMV isolates.

Phytochemical profile of petals from black Dahlia pinnata by flow injection analysis-electrospray ionization-Fourier transform ion cyclotron resonance mass spectrometry.

INTRODUCTION: Dahlia pinnata Cav. is a flower native to Mexico that has many applications; in particular, its petals have been used for ornamental, food, and medicinal purposes, for example to treat skin rashes and skin cracks. It has been reported that the medicinal properties of plants are generally related to the phytochemical constituents they possess. However, there are few studies on black D. pinnata. OBJECTIVES: The present study was aimed at qualitatively and quantitatively determining the phytochemical profile of petals from black D. pinnata. METHODOLOGY: Phytochemicals from Dahlia petals were extracted by consecutive maceration (hexane, dichloromethane, and methanol); then, the extracts were analyzed through colorimetric assays and UV-Vis spectroscopy for qualitative identification and quantification of phytochemical compounds, respectively. The methanolic extract was analyzed by flow injection analysis-electrospray ionization-Fourier transform ion cyclotron resonance mass spectrometry (FIA-ESI-FTICR-MS) in negative and positive mode. RESULTS: Quantitative phytochemical profiling of the methanolic extract by UV-Vis spectroscopy indicated high contents of phenolic compounds (34.35 ± 3.59 mg EQ/g plant) and sugars (23.91 ± 1.99 mg EQ/g plant), while the qualitative profiling by FIA-ESI-FTICR-MS allowed the tentative identification of several flavonoids and phenolic acids. Kaempferol-3-rutinoside, pelargonidin-3-(6″-malonylglucoside)-5-glucoside, rutin, kaempferol-3-(2″,3″-diacetyl-4″-p-coumaroylrhamnoside), and myricetin-3-(2‴-galloylrhamnoside) were the main compounds detected. CONCLUSION: The results expand our knowledge of the phytochemical constituents of petals from black D. pinnata.

[Pathogenicity and induced systemic resistance of Fusarium oxysporum and Verticillium dahlia to Salvia miltiorrhiza].

Salvia miltiorrhiza is a commonly used bulk medicinal material in China. Due to the increasing demand in recent years, the planting area is expanding. In the artificial cultivation of S. miltiorrhiza, continuous cropping obstacles are prominent, which has seriously restrained the growth of S. miltiorrhiza, resulted in serious root diseases, and affected the yield and quality of medicinal materials. The pathogen infection can induce plant resistance. Previously, this research group isolated Fusarium oxysporum and Verticillium dahlia from the roots of diseased S. miltiorrhiza. In this study, 7 days after inoculation of S. miltiorrhiza with F. oxysporum(Foc group) and V. dahlia(Vd group), the incidence rates in S. miltiorrhiza were 48% and 26%, respectively. Both the two pathogens significantly reduced the aboveground biomass of S. miltiorrhiza. Five days after inoculation, the activities of defensive enzymes, such as peroxidase(POD), phenylalanine ammonia-lyase(PAL), superoxide dismutase(SOD), and polyphenol oxidase(PPO) reached the peak. The enzyme activity of the Foc group was significantly higher than that of the Vd group. Three days after inoculation, the expression of defense genes SmPDF2.1 and SmPR10 peaked and then decreased. The results showed that F. oxysporum and V. dahlia showed pathogenicity to S. miltiorrhiza and could strongly induce systemic resistance. In terms of the above indexes, F. oxysporum was superior to V. dahlia.

Comparison of two highly sensitive methods to detect potato spindle tuber viroid in Dahlia using quantitative-reverse transcription-polymerase chain reaction assays.

Potato spindle tuber viroid (PSTVd) belongs to the Pospiviroidae family and is the type species for the genus Pospiviroid. In 2011, PSTVd was first detected in dahlias in Japan. Since that time, unregistered PSTVd isolates have been identified in seven field-grown dahlia cultivars. None of the infected dahlias showed disease symptoms during the early stages of infection, however, growth suppression occasionally occurred during later stages. Therefore, in dahlia, diagnosing PSTVd by the external appearance of plants is difficult, and the threat of new PSTVd isolates spreading to other susceptible hosts still remains. In this study, we developed an efficient inspection method using several dahlia plant tissues and organs including dried bulbs. This developed method will be useful for inspecting seedlings to prevent the invasion of PSTVd at the border.

Identification and Molecular Mechanisms of Key Nucleotides Causing Attenuation in Pathogenicity of Dahlia Isolate of Potato Spindle Tuber Viroid.

While the potato spindle tuber viroid (PSTVd) variant, PSTVd-Dahlia (PSTVd-D or PSTVd-Dwt) induces very mild symptoms in tomato cultivar 'Rutgers', PSTVd-Intermediate (PSTVd-I or PSTVd-Iwt) induces severe symptoms. These two variants differ by nine nucleotides, of which six mutations are located in the terminal left (TL) to the pathogenicity (P) domains. To evaluate the importance of mutations located in the TL to the P domains, ten types of point mutants were created by swapping the nucleotides between the two viroid variants. Bioassay in tomato plants demonstrated that two mutants created on PSTVd-Iwt at positions 42 and 64 resulted in symptom attenuation. Phenotypic and RT-qPCR analysis revealed that mutation at position 42 of PSTVd-Iwt significantly reduced disease severity and accumulation of the viroid, whereas mutation at position 64 showed a significant reduction in stunting when compared to the PSTVd-Iwt infected plant. RT-qPCR analysis on pathogenesis-related protein 1b1 and chalcone synthase genes showed a direct correlation with symptom severity whereas the expansin genes were down-regulated irrespective of the symptom severity. These results indicate that the nucleotides at positions 42 and 64 are in concert with the ones at positions 43, 310, and 311/312, which determines the slower and stable accumulation of PSTVd-D without eliciting excessive host defense responses thus contributing in the attenuation of disease symptom.

Edible flowers as sources of phenolic compounds with bioactive potential.

The edible flowers are widely used, but there is still a lot to be done in relation to its bioactive potential and its correlation with the presence of phenolic compounds. The aim of this study was determined the individual phenolic profile in the hydromethanolic extracts and infusion preparations of four different flower samples (Dahlia mignon, Rosa damascena 'Alexandria' and R. gallica 'Francesa' draft in R. canina, Calendula officinalis L., and Centaurea cyanus L.) and their bioactive potential (antioxidant, antiproliferative, and antibacterial capacity). All the studied flowers presented different profiles regarding their phenolic composition and revealed biological potential. The bioactive potential of the studied flowers was moderate, the hydromethanolic extracts of rose petals showed the best results for antioxidant and antibacterial assays, while the antiproliferative properties were only present in some of the tested cell lines, for the hydromethanolic extracts, in which dahlia and rose showed the best results. These results demonstrate that edible flowers can be used as a source of phenolic compounds with bioactive potential, which can be applied in the food sector, as foods and as sources natural ingredients.

Application of dahlia-like molybdenum disulfide nanosheets for solid phase extraction of Co(II) in vegetable and water samples.

Recently, molybdenum disulfide has been attracted considerable attention due to its unique three layered structure. Adsorption sites with abundant electron density on edges and surfaces of MoS2 can adsorb different metal ions with no need to ligand and functionalization. In this study, dahlia-like MoS2 nanosheets were prepared by a simple hydrothermal method and characterized using different tools such as FESEM, TEM, EDX, XRD, DLS and zeta potential measurements. Then, they were applied for solid phase extraction of Co(II) as an example of heavy metals. Different factors (the pH, adsorbent amount, contact time, type of eluent, matrix and reusability) affecting the extraction process were studied. Under optimum conditions, the relative standard deviation, adsorption capacity and limit of detection were 2.3%, 80.0mgg-1 and 0.31µgL-1, respectively. The accuracy of the method was confirmed by analyzing the standard reference material (SRM 1640a) and spiked real samples.

Nutritional and chemical characterization of edible petals and corresponding infusions: Valorization as new food ingredients.

Edible flowers provide new colours, textures and vibrancy to any dish, and apart from the "glam" factor, they can constitute new sources of bioactive compounds. In the present work, the edible petals and infusions of dahlia, rose, calendula and centaurea, were characterized regarding their nutritional value and composition in terms of hydrophilic and lipophilic compounds. Carbohydrates were the most abundant macronutrients, followed by proteins and ash. Fructose, glucose and sucrose were identified in all the petals and infusions. Rose petals and calendula infusions gave the highest content of organic acids, mainly due to the presence of malic and quinic acids, respectively. Polyunsaturated fatty acids predominated over saturated fatty acids, mainly due to the contribution of linoleic acid. Calendula presented the highest content in tocopherols, with α-tocopherol as the most abundant. These results highlight the interest of edible petals "as" and "in" new food products, representing rich sources of bioactive nutrients.

Production of inulinase, fructosyltransferase and sucrase from fungi on low-value inulin-rich substrates and their use in generation of fructose and fructo-oligosaccharides.

Owing to applications in the food and nutraceutical industries, inulinases, fructosyltransferases and sucrases have gained considerable attention in recent times. Twenty-five fungal strains were screened for production of these enzymes on three different media formulated using inulin-rich plant extracts prepared from asparagus root, dahlia tuber and dandelion root extract. Culture filtrates of the fungi were examined for hydrolytic activities. Fungi belonging to genus Aspergillus, A. niger GNCC 2655 (11.3 U/ml), A. awamori MTCC 2879 (8.2 U/ml), A. niger ATCC 26011 (7.9 U/ml) secreted high titers of inulinase followed by Penicillium sp. NFCCI 2768 (2.6 U/ml) and Penicillium citrinum MTCC 1256 (1.1 U/ml). High sucrase activity was noticed in A. niger GNCC 2613 (113 U/ml) and A. awamori MTCC 2879 (107.8 U/ml). Analysis of end products of inulinase action by HPLC revealed that most of the enzymes were exo-inulinases liberating fructose exclusively from inulin. Five fungi, P. citrinum MTCC 1256, Penicillium rugulosum MTCC 3487, Penicillium sp. NFCCI 2768, A. fumigatus GNCC 1351 and A. niger ATCC 26011 however, produced a mixture of endo- and exo-inulinases liberating oligosaccharides (GF3 and GF2) along with fructose. High inulinase/sucrase yielding strains were evaluated for extracellular and intracellular hydrolytic and transfructosylating activities and intracellular enzyme profiles were found to be considerably different in terms of titers and end products.

Identification of insecticidal constituents of the essential oils of Dahlia pinnata Cav. against Sitophilus zeamais and Sitophilus oryzae.

The aim of this research was to determine the chemical composition of the essential oils of Dahlia pinnata, their insecticidal activity against Sitophilus zeamais and Sitophilusoryzae and to isolate insecticidal constituents. Based on bioactivity-guided fractionation, active constituents were isolated and identified as D-limonene, 4-terpineol and α-terpineol. Essential oils and active compounds tested exhibited contact toxicity, with LD50 values ranging from 132.48 to 828.79 µg/cm(2) against S. zeamais and S. oryzae. Essential oils possessed fumigant toxicity against S. zeamais and S. oryzae with LC50 from 14.10 to 73.46 mg/L. d-Limonene (LC50 = 4.55 and 7.92 mg/L) showed stronger fumigant toxicity against target insects. 4-Terpineol (88 ± 8%) and d-limonene (87 ± 5%) showed the strongest repellency against S. zeamais and S. oryzae, respectively. The results indicate that essential oils and insecticidal constituents have potential for development into natural fumigants, insecticides or repellents for control of the stored-product insect pests.

Production of inulinase from Kluyveromyces marxianus using Dahlia tuber extract

Various carbon sources were evaluated for production of inulinase by yeast, Kluyveromyces marxianus MTCC 3995. Highest inulinase activity was observed with Dahlia extract (25.3 nkat mL-1) as carbon source. The enzyme activity was 1.4 folds higher than that observed in media containing pure chicory inulin (17.8 nkat mL-1). The yeast showed good growth on a simple medium containing dahlia extract (20% w/v) and yeast extract (2%w/v) as carbon and nitrogen source respectively, in 96 h. at 28°C and 120 rpm. Lowest inulinase yield (4.8 nkat mL-1) was seen in the medium containing glucose as C-source. Although varied inulinase levels were noticed on different C- sources, Inulinase: Sucrase (I/S) ratios were noticed to be similar. Among various protein sources tested, yeast extract was found to be the best source followed by beef extract (17.9 nkat mL-1) and peptone (13.8 nkat mL-1). The enzyme was optimally active at pH (4.0) and 50°C. TLC analysis of end product revealed that inulinase hydrolyzed inulin exclusively into fructose. Results suggest that the dahlia extract induced exoinulinase synthesis in Kluyveromyces marxianus and can be utilized as a potential substrate for inulinase production.

Fructo-oligosaccharide production from inulin through partial citric or phosphoric acid hydrolyses.

Purified inulin from Dahlia tubers was partially hydrolyzed to form fructo-oligosaccharides by using citric or phosphoric acids (pH, 2.0-2.5) as mild acid catalysts. The ideal kinetic conditions to ensure a high yield of fructo-oligosaccharides relative to free fructose were a temperature range of 85°C-95°C, a hydrolysis time of 15-25 minutes, and a catalyst pH of 2.5. At the higher temperature and the longest hydrolysis time, an inversion of the product ratio occurred. Under these conditions, co-generation of hydroxymethylfurfural occurred, and it was eliminated by activated charcoal. Unlike in classic hydrolysis with hydrochloric or sulfuric acid, deionization of the actual hydrolysates was not necessary because the catalyst neutralization with common bases results in the formation of co-nutrients with alternative uses as foods or fermentation substrates. These whole hydrolysates can be advantageously added as nutraceuticals to carbonated beverages and acidic foods, such as soft drinks and yogurts.

Delta inulin: a novel, immunologically active, stable packing structure comprising ß-D-[2 -> 1] poly(fructo-furanosyl) α-D-glucose polymers.

We report a novel isoform of ß-D-[2 → 1] poly(fructo-furanosyl) α-D-glucose termed delta inulin (DI), comparing it with previously described alpha (AI), beta (BI) and gamma (GI) isoforms. In vitro, DI is the most immunologically active weight/weight in human complement activation and in binding to monocytes and regulating their chemokine production and cell surface protein expression. In vivo, this translates into potent immune adjuvant activity, enhancing humoral and cellular responses against co-administered antigens. As a biocompatible polysaccharide particle, DI is safe and well tolerated by subcutaneous or intramuscular injection. Physico-chemically, DI forms as an insoluble precipitate from an aqueous solution of suitable AI, BI or GI held at 37-48°C, whereas the precipitate from the same solution at lower temperatures has the properties of AI or GI. DI can also be produced by heat conversion of GI suspensions at 56°C, whereas GI is converted from AI at 45°C. DI is distinguished from GI by its higher temperature of solution in dilute aqueous suspension and by its lower solubility in dimethyl sulfoxide, both consistent with greater hydrogen bonding in DI's polymer packing structure. DI suspensions can be dissolved by heat, re-precipitated by cooling as AI and finally re-converted back to DI by repeated heat treatment. Thus, DI, like the previously described inulin isoforms, reflects the formation of a distinct polymer aggregate packing structure via reversible noncovalent bonding. DI forms the basis for a potent new human vaccine adjuvant and further swells the growing family of carbohydrate structures with immunological activity.