In vitro and in vivo antimicrobial efficacy of essential oils and individual compounds against Phytophthora parasitica var. nicotianae

Aim

To evaluate the antimicrobial effects of essential oils (EOs) from cassia, basil, geranium, lemongrass, cumin and thyme, as well as their major components, against Phytophthora parasitica var. nicotianae; to investigate morphological changes in hyphae and sporangia in response to treatment with cinnamaldehyde; and to further evaluate potential biocontrol capacities against tobacco black shank under greenhouse conditions.

Methods and Results

The results revealed that the extent of mycelial growth inhibition was primarily dependent on the composition and concentration of the EOs and the structure of individual compounds. Cinnamaldehyde had a significantly higher inhibitory effect on mycelial growth, formation of sporangia, and production and germination of zoospores in P. parasitica var. nicotianae in vitro, achieving complete inhibition of these phenotypes at 72, 36, 36 and 18 mg l^?1, respectively. Scanning electron microscopic observations revealed that cinnamaldehyde can cause considerable morphological degenerations of hyphae and sporangia such as cytoplasmic coagulation, shrivelled mycelia and sporangia aggregates and swelling and lysis of mycelia and sporangia walls. In vivo assays with cinnamaldehyde demonstrated that this compound afforded protective effect against tobacco black shank under greenhouse conditions in susceptible tobacco plants.

Conclusions

The results of in vitro and in vivo bioassays, together with SEM imaging of the microstructure of P. parasitica var. nicotianae supported the possibility of using cinnamaldehyde as a potent natural biofungicide in the greenhouse.

Significance and Impact of the Study

This study provides a theoretical basis for the potential use of cinnamaldehyde as commercial agents or lead compounds that can be exploited as commercial biofungicides in the protection of tobacco plants from P. parasitica var. nicotianae infection.     

Factors restraining parasitism of the invasive vine Mikania micrantha by the holoparasitic plant Cuscuta campestris

Mikania micrantha (Asteraceae) is one of the 10 most invasive weeds in the world and has caused tremendous economic and environmental losses in southern China. The dodder Cuscuta campestris (Convolvulaceae) is a native holoparasite that can parasitize and suppress M. micrantha, and thus is recommended as an effective control agent. However, the natural growth of dodder lags behind that of M. micrantha and fails to exert direct year-round suppression. To verify the effective parasitic distance, we placed a dodder seedling at a designated distance from the stem of M. micrantha and monitored coiling, haustorium formation, and survival. To verify suitable host stems for the parasite, we grew M. micrantha for more than 6 months to form stems of different sizes, taped dodder seedlings to the stems, and monitored. We used various temperatures to determine the effect on dodder seed germination and M. micrantha sprouting. The results showed that a dodder–host distance of 4 cm decreased the probability of successful parasitism to 0; dodder seedlings cannot parasitize M. micrantha stem diameters ≥0.3 cm; and the temperatures for the highest dodder seed germination and M. micrantha sprouting are 26 and 30 °C, respectively. We conclude that the lack of suitable M. micrantha parts within the dodder’s effective parasitism distance is the major cause of restricted dodder parasitism and that the lower temperature for the highest dodder germination compared to that for the highest M. micrantha sprouting may decrease the possibility of parasitism. To acquire aggressive parasitism, dodder should be manually assisted by dispersing its vegetative form.     

Net greenhouse gas balance in response to nitrogen enrichment: perspectives from a coupled biogeochemical model

Increasing reactive nitrogen (N) input has been recognized as one of the important factors influencing climate system through affecting the uptake and emission of greenhouse gases (GHG). However, the magnitude and spatiotemporal variations of N‐induced GHG fluxes at regional and global scales remain far from certain. Here we selected China as an example, and used a coupled biogeochemical model in conjunction with spatially explicit data sets (including climate, atmospheric CO₂, O₃, N deposition, land use, and land cover changes, and N fertilizer application) to simulate the concurrent impacts of increasing atmospheric and fertilized N inputs on balance of three major GHGs (CO₂, CH₄, and N₂O). Our simulations showed that these two N enrichment sources in China decreased global warming potential (GWP) through stimulating CO₂ sink and suppressing CH₄ emission. However, direct N₂O emission was estimated to offset 39% of N‐induced carbon (C) benefit, with a net GWP of three GHGs averaging ?376.3 ± 146.4 Tg CO₂ eq yr^?1 (the standard deviation is interannual variability of GWP) during 2000–2008. The chemical N fertilizer uses were estimated to increase GWP by 45.6 ± 34.3 Tg CO₂ eq yr^?1 in the same period, and C sink was offset by 136%. The largest C sink offset ratio due to increasing N input was found in Southeast and Central mainland of China, where rapid industrial development and intensively managed crop system are located. Although exposed to the rapidly increasing N deposition, most of the natural vegetation covers were still showing decreasing GWP. However, due to extensive overuse of N fertilizer, China's cropland was found to show the least negative GWP, or even positive GWP in recent decade. From both scientific and policy perspectives, it is essential to incorporate multiple GHGs into a coupled biogeochemical framework for fully assessing N impacts on climate changes.     

Bioconversion of Agave tequilana fructans by exo-inulinases from indigenous Aspergillus niger CH-A-2010 enhances ethanol production from raw Agave tequilana juice

Agave tequilana fructans are the source of fermentable sugars for the production of tequila. Fructans are processed by acid hydrolysis or by cooking in ovens at high temperature. Enzymatic hydrolysis is considered an alternative for the bioconversion of fructans. We previously described the isolation of Aspergillus niger CH-A-2010, an indigenous strain that produces extracellular inulinases. Here we evaluated the potential application of A. niger CH-A-2010 inulinases for the bioconversion of A. tequilana fructans, and its impact on the production of ethanol. Inulinases were analyzed by Western blotting and thin layer chromatography. Optimal pH and temperature conditions for inulinase activity were determined. The efficiency of A. niger CH-A-2010 inulinases was compared with commercial enzymes and with acid hydrolysis. The hydrolysates obtained were subsequently fermented by Saccharomyces cerevisiae to determine the efficiency of ethanol production. Results indicate that A. niger CH-A-2010 predominantly produces an exo-inulinase activity. Optimal inulinase activity occurred at pH 5.0 and 50 °C. Hydrolysis of raw agave juice by CH-A-2010 inulinases yielded 33.5 g/l reducing sugars, compared with 27.3 g/l by Fructozyme^® (Novozymes Corp, Bagsværd, Denmark) and 29.4 g/l by acid hydrolysis. After fermentation of hydrolysates, we observed that the conversion efficiency of sugars into ethanol was 97.5 % of the theoretical ethanol yield for enzymatically degraded agave juice, compared to 83.8 % for acid-hydrolyzed juice. These observations indicate that fructans from raw Agave tequilana juice can be efficiently hydrolyzed by using A. niger CH-A-2010 inulinases, and that this procedure impacts positively on the production of ethanol.     

Genetic and physical fine mapping of a multilocular gene Bjln1 in Brassica juncea to a 208-kb region

Most of the germplasm resources in Brassica juncea produce silique with only two locules, whereas a few varieties can produce silique with three or four locules. The increase in locule number in B. juncea has been shown to cause an increase in the number of seeds per silique, resulting in an increase in the yield per plant. Thus, the development of high-locule-number varieties may be an effective way of improving the yield of B. juncea. Duoshi, a B. juncea landrace originating from the Qinghai–Tibetan plateau, produces silique with 3–4 locules. Genetic analysis has shown that the high-locule-number trait in Duoshi is determined by two recessive genes, tentatively designated as Bjln1 and Bjln2. For fine mapping of the Bjln1 gene, a BC3 population was developed from the cross between Duoshi (multilocular parent) and Xinjie (bilocular parent). Using a combination of amplified fragment length polymorphism (AFLP) and bulked segregant analysis, only two AFLP markers linked to Bjln1 were identified. Preliminary linkage analysis showed that the two AFLP markers were located on the same side of Bjln1. Blast analysis revealed that the sequences of the two AFLP markers had homologues on Scaffold000019 at the bottom of B. rapa A7. Using the results of linkage analysis and BlastN searches, simple sequence repeat (SSR) markers were subsequently developed based on the sequence information from B. rapa A7. Seven SSR markers were eventually identified, of which ln 8 was co-segregated with Bjln1. ln 7 and ln 9, the closest flanking markers, were mapped at 2.0 and 0.4 cM distant from the Bjln1 gene, respectively. The SSR markers were cloned, sequenced and mapped on A7 of B. rapa (corresponding to J7 in the A genome of B. juncea). The two closest flanking markers, ln 7 and ln 9, were mapped within a 208-kb genomic region on B. rapa A7, in which the Bjln1 gene might be included. The present study may facilitate cloning of the Bjln1 gene as well as the selection process for developing multilocular varieties in B. juncea by marker-assisted selection and genetic engineering.     

Novel Systems for Dynamically Assessing Insulin Action in Live Cells Reveals Heterogeneity in the Insulin Response

Regulated GLUT4 trafficking is a key action of insulin. Quantitative stepwise analysis of this process provides a powerful tool for pinpointing regulatory nodes that contribute to insulin regulation and insulin resistance. We describe a novel GLUT4 construct and workflow for the streamlined dissection of GLUT4 trafficking; from simple high throughput screens to high resolution analyses of individual vesicles. We reveal single cell heterogeneity in insulin action highlighting the utility of this approach – each cell displayed a unique and highly reproducible insulin response, implying that each cell is hard‐wired to produce a specific output in response to a given stimulus. These data highlight that the response of a cell population to insulin is underpinned by extensive heterogeneity at the single cell level. This heterogeneity is pre‐programmed within each cell and is not the result of intracellular stochastic events.     

The role of glycogen synthase kinase-3β in glioma cell apoptosis induced by remifentanil

The aim of malignant glioma treatment is to inhibit tumor cell proliferation and induce tumor cell apoptosis. Remifentanil is a clinical anesthetic drug that can activate the N-methyl-D-aspartate (NMDA) receptor. NMDA receptor signaling activates glycogen synthase kinase-3β (GSK-3β). Discovered some 32 years ago, GSK-3β was only recently considered as a therapeutic target in cancer treatment. The purpose of this study was to assess whether remifentanil can induce the apoptosis of C6 cells through GSK-3β activation. 3-(4,5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) was used to detect cell viability. Hoechst 33342 staining and flow cytometry were used to detect cell apoptosis. The effect of GSK-3β activation was detected using a GSK-3β activation assay kit and 4-benzyl-2-methyl-1,2,4-thiadiazolidine-3,5-dione (TDZD-8), a potent and selective small molecule inhibitor of GSK-3β. The MTT assay indicated that remifentanil induced C6 cell death in a concentration- and time-dependent manner. Hoechst 33342 staining and flow cytometry showed that remifentanil significantly induced C6 cell apoptosis. The measurement of GSK-3β activation showed that remifentanil increased the cellular level of GSK-3β. All of these toxic effects can be attenuated by treatment with TDZD-8. These results suggest that remifentanil is able to induce C6 cell apoptosis through GSK-3β activation, which provides a basis for its potential use in the treatment of malignant gliomas.     

Nitrate signals determine the sensing of nitrogen through differential expression of genes involved in nitrogen uptake and assimilation in finger millet

In order to understand the molecular basis of high nitrogen use efficiency of finger millet, five genes (EcHNRT2, EcLNRT1, EcNADH-NR, EcGS, and EcFd-GOGAT) involved in nitrate uptake and assimilation were isolated using conserved primer approaches. Expression profiles of these five genes along with the previously isolated EcDof1 was studied under increased KNO₃ concentrations (0.15 to 1,500 μM) for 2 h as well as at 1.5 μM for 24 h in the roots and shoots of 25 days old nitrogen deprived two contrasting finger millet genotypes (GE-3885 and GE-1437) differing in grain protein content (13.76 and 6.15 %, respectively). Time kinetics experiment revealed that, all the five genes except EcHNRT2 in the leaves of GE-3885 were induced within 30 min of nitrate exposure indicating that there might be a greater nitrogen deficit in leaves and therefore quick transportation of nitrate signals to the leaves. Exposing the plants to increasing nitrate concentrations for 2 h showed that in roots of GE-3885, NR was strongly induced while GS was repressed; however, the pattern was found to be reversed in leaves of GE-1437 indicating that in GE-3885, most of the nitrate might be reduced in the roots but assimilated in leaves and vice-versa. Furthermore, compared with the low-protein genotype, expression of HNRT2 was strongly induced in both roots and shoots of high-protein genotype at the least nitrate concentration supplied. This further indicates that GE-3885 is a quick sensor of nitrogen compared with the low-protein genotype. Furthermore, expression of EcDof1 was also found to overlap the expression of NR, GS, and GOGAT indicating that Dof1 probably regulates the expression of these genes under different conditions by sensing the nitrogen fluctuations around the root zone.     

Subtype Identification in Acutely Dissociated Rat Nodose Ganglion Neurons Based on Morphologic Parameters

Nodose ganglia are composed of A-, Ah- and C-type neurons. Despite their important roles in regulating visceral afferent function, including cardiovascular, pulmonary, and gastrointestinal homeostasis, information about subtype-specific expression, molecular identity, and function of individual ion transporting proteins is scarce. Although experiments utilizing the sliced ganglion preparation have provided valuable insights into the electrophysiological properties of nodose ganglion neuron subtypes, detailed characterization of their electrical phenotypes will require measurements in isolated cells. One major unresolved problem, however, is the difficulty to unambiguously identify the subtype of isolated nodose ganglion neurons without current-clamp recording, because the magnitude of conduction velocity in the corresponding afferent fiber, a reliable marker to discriminate subtypes in situ, can no longer be determined. Here, we present data supporting the notion that application of an algorithm regarding to microscopic structural characteristics, such as neuron shape evaluated by the ratio between shortest and longest axis, neuron surface characteristics, like membrane roughness, and axon attachment, enables specific and sensitive subtype identification of acutely dissociated rat nodose ganglion neurons, by which the accuracy of identification is further validated by electrophysiological markers and overall positive predictive rates is 89.26% (90.04%, 76.47%, and 98.21% for A-, Ah, and C-type, respectively). This approach should aid in gaining insight into the molecular correlates underlying phenotypic heterogeneity of nodose ganglia. Additionally, several critical points that help for neuron identification and afferent conduction calibration are also discussed.