Evaluating type III polyketide synthase (PKS) and terpene synthase (TPS) expression in incompatible interactions of Zingiber zerumbet to Pythium myriotylum Drechsler

Abstract

Pythium species are aggressive soil-borne necrotrophic oomycetes causing soft-rot disease of ginger. Disease severity indices determined following infection with P. myriotylum Drechsler in ginger cultivar, Zingiber officinale cv. Varada and a wild congener, Z. zerumbet at varying zoospore concentrations (10⁴–10¹² spores/ml) revealed high disease severity (100%) in ginger cultivar whereas Z. zerumbet displayed resistance. Absence of positive correlation between Z. zerumbet resistance and polyphenolic content indicates role of polyketides and zerumbone in preventing pathogen ingress, as reported earlier. Towards elucidating this, Z. zerumbet specific polyketide synthase (PKS) and terpene synthase (TPS) gene sequences designated ZzTPS and ZzPKS respectively were characterised. Phylogenetic analysis clustered ZzTPS with TPS-b sub-family and ZzPKS with non-chalcone forming PKS. ZzTPS and ZzPKS showed biphasic expression with first at 6?hours post infection (hpi) and then at 8 hpi, indicative of rapid induction followed by reinforcement to sustain resistance mechanisms in the wild taxon.     

Biocontrol activity of the extract prepared from Zingiber zerumbet for the management of rhizome rot in Zingiber officinale caused by Pythium myriotylum

Zingiber zerumbet Smith or wild ginger is remarkable for its inherent resistance to Pythium spp., which cause soft rot disease in Zingiber officinale Rosc. In the present study, various concentrations of extract prepared from Z. zerumbet were screened for its activity against Pythium myriotylum. Microscopic observation of P. myriotylum in presence of Z. zerumbet extract has confirmed the complete lysis of pathogen within 10 h. However, the same treatment with Z. officinale extract was found to have partial antifungal effect even after 24 h due to inability of its metabolites to prevent the growth of P. myriotylum. Due to the antifungal activity, extract from Z. zerumbet was subjected to GC–MS and LC-QTOF-MS which has identified Zerumbone with m/z 219 as the major compound. Further, in vivo study and the subsequent microscopic analysis have confirmed the applicability of extract from Z. zerumbet as a phytomedicine to control rhizome rot in ginger.     

Chemical Composition,Antimicrobial and Insecticidal Activities of Flowers Essential Oils of Alpinia zerumbet (Pers.) B.L.Burtt & R.M.Sm. from Martinique Island

Alpinia species, used as ornamental plants and flavoring agents, are renowned for their therapeutic properties and their subsequent use in traditional medicines throughout the world. Alpinia zerumbet (Pers .) B.L.Burtt & R.M.Sm . is the most common Alpinia species encountered in Martinique. Several essential oils (EOs) obtained by hydrodistillation of A. zerumbet flowers collected in various locations on the island at different seasons were analysed to evaluate the influence of the collection period and area on the EOs’ chemical compositions and to assess their bioactivity. A combination of GC‐FID and GC/MS techniques was used to examine the volatile constituents, leading to the identification of a total of 71 components accounting for 97.8 – 99.3% of the respective total GC‐FID areas: among them, nineteen compounds were characterized for the first time in A. zerumbet EOs. The antimicrobial activity of these EOs was assessed against eight bacterial, yeast and fungal strains and two multi‐resistant strains: some significant bacteriostatic and fungistatic activities of A. zerumbet flower oils were evidenced. Finally, an interesting insecticidal activity of the flower EO was highlighted for the first time against Aedes aegypti.     

Chemical carcinogens in non-enzymatic cytosine deamination: 3-isocyanatoacrylonitrile

Uracil has long been known as the main product of nitrosative cytosine deamination in aqueous solution. Recent mechanistic studies of cytosinediazonium ion suggest that the cation formed by its dediazoniation can ring-open to N-protonated (Z,s-cis)-3-isocyanatoacrylonitrile 7. Stereochemical preferences are discussed of the 3-isocyanatoacrylonitriles (Z,s-cis)-10, (E,s-cis)-11, (Z,s-trans)-12, and (E,s-trans)-13. The electronic structures of 7 and 10–13 have been analyzed and a rationale is provided for the thermodynamic preference for (Z,s-cis)-10. It is shown that s-cis/s-trans-interconversion occurs via C−N rotation–inversion paths with barriers below 3 kcal mol⁻¹. The proton affinities of 3-isocyanatoacrylonitrile 10 and water are nearly identical and, thus, 3-isocyanatoacrylonitriles can and should be formed in aqueous media from 7 along with 3-aminoacrylonitriles 9. The results highlight the relevance of the chemistry of 3-isocyanatoacrylonitriles for the understanding of the chemical toxicology of nitrosation of the nucleobase cytosine. Electronic Supplementary Material Supplementary Material is available for this article at Dedicated to professor Dr. Paul von Ragué Schleyer on the occasion of his 75th birthday     

Molecular phylogenetics and anti-<Emphasis Type="Italic">Pythium</Emphasis> activity of endophytes from rhizomes of wild ginger congener, <Emphasis Type="Italic">Zingiber zerumbet</Emphasis> Smith

Zingiber zerumbet, a perennial rhizomatous herb exhibits remarkable disease resistance as well as a wide range of pharmacological activities. Towards characterizing the endophytic population of Z. zerumbet rhizomes, experiments were carried out during two different growing seasons viz., early-June of 2013 and late-July of 2014. A total of 34 endophytes were isolated and categorized into 11 morphologically distinct groups. Fungi were observed to predominate bacterial species with colonization frequency values ranging from 12.5 to 50 %. Among the 11 endophyte groups isolated, molecular analyses based on ITS/16S rRNA gene sequences identified seven isolate groups as Fusarium solani, two as F. oxysporum and one as the bacterium Rhizobium spp. Phylogenetic tree clustered the ITS sequences from Z. zerumbet endophytes into distinct clades consistent with morphological and sequence analysis. Dual culture assays were carried out to determine antagonistic activity of the isolated endophytes against Pythium myriotylum, an economically significant soil-borne phytopathogen of cultivated ginger. Experiments revealed significant P. myriotylum growth inhibition by F. solani and F. oxysporum isolates with percentage of inhibition (PoI) ranging from 45.17 ± 0.29 to 62.2 ± 2.58 with F. oxysporum exhibiting higher PoI values against P. myriotylum. Using ZzEF8 metabolite extract, concentration-dependent P. myriotylum hyphal growth inhibition was observed following radial diffusion assays. These observations were confirmed by scanning electron microscopy analysis wherein exposure to ZzEF8 metabolite extract induced hyphal deformities. Results indicate Z. zerumbet endophytes as promising resources for biologically active compounds and as biocontrol agents for soft rot disease management caused by Pythium spp.     

When and where was the most recent common ancestor?

 The structured coalescent is investigated for single-locus, digenic samples in the diffusion limit of the unidimensional stepping-stone model for homogeneous, isotropic migration and random genetic drift. Let T denote the scaled time to the most recent common ancestor (MRCA) of the two genes, and let Z designate the scaled deviation of the position of the MRCA from the average position of the two genes. The joint probability density of T and Z is evaluated explicitly. Both the marginal and conditional distributions of T have infinite expectation, as does the marginal distribution of Z. Conditioned on T = τ, the distribution of Z is Gaussian with mean zero and variance 2τ. The main results are extended to anisotropic migration. The results establish the existence of and define in the diffusion limit a retrospective stochastic process for digenic samples in one spatial dimension. Received: 1 May 2001 / Revised version: 2 September 2001 / Published online: 8 February 2002     

Substrate specificity of α‐humulene synthase from Zingiber zerumbet Smith and determination of kinetic constants by a spectrophotometric assay

Terpene synthases are the key enzymes in terpene biosynthesis that provide a structurally complex and highly diverse product spectrum. A suitable and reliable analytical assay is indispensable to measure terpene synthase activity accurately and precisely. In this study, a malachite green assay (MG) was adapted to rapidly assay terpene synthase activity and was validated in comparison to an already established gas chromatography assay. A linear correlation between both assays was observed. Kinetic properties for the previously described sesquiterpene synthase α‐humulene synthase (HUM) from Zingiber zerumbet Smith were investigated for the bioconversion of the monoterpene precursors geranyl pyrophosphate (2E‐GPP) and neryl pyrophosphate (2Z‐NPP) as well as for the sesquiterpene precursor farnesyl pyrophosphate (2E,6E‐FPP). Also, gas chromatography mass spectrometry (GS‐MS) was carried out to identify the products of the bioconversion of (2E)‐GPP and (2Z)‐NPP.     

Molecular Characterization of an Oomycete-Responsive PR-5 Protein Gene from <Emphasis Type="Italic">Zingiber zerumbet</Emphasis>

The tropical spice crop ginger (Zingiber officinale Roscoe) is highly susceptible to soft rot disease caused by the necrotrophic oomycete Pythium aphanidermatum (Edson) Fitzp. However, Zingiber zerumbet (L.) Smith, a wild relative of ginger, is resistant to P. aphanidermatum and has been proposed as a potential donor for soft rot resistance to Z. officinale. We identified a member of the pathogenesis-related protein group 5 (PR-5) gene family in Z. zerumbet that is expressed constitutively but upregulated in response to infection by P. aphanidermatum. Expression of this gene was upregulated as early as 1.5 h post inoculation (hpi) with the pathogen, peaked at 6 hpi, declined by 9 hpi, and again peaked at 15 hpi before declining at 48 hpi. A cDNA of this PR-5 gene, designated as ZzPR5, encodes a 226-amino-acid predicted protein with a calculated pI of 5.05. The N terminus of this protein contains a 22-amino-acid signal peptide, suggesting that the protein may show apoplastic accumulation like other acidic PR-5 proteins. Phylogenetic analysis revealed high similarity between ZzPR5 and PR-5 proteins reported from other plant species, especially from other Zingiberales. Molecular modeling of ZzPR5 protein revealed an acidic surface cleft, a feature characteristic of glycoside hydrolases and antifungal PR-5 proteins. In molecular docking studies, a linear polymeric molecule of (1,3)-β-d-glucan, a major constituent of the oomycete cell wall, fitted favorably into the surface cleft of ZzPR5 and interacted with acidic amino acids known to be involved in glucan hydrolysis, suggesting a potential antioomycete activity for ZzPR5 protein. Elucidation of the molecular mechanism of ZzPR5 may provide important insight toward engineering soft rot resistance into the obligatory asexual ginger.     

Towards genetic improvement of commercially important microalga Haematococcus pluvialis for biotech applications

Haematococcus pluvialis is a unicellular green alga which produces a ketocarotenoid, astaxanthin which has pharmaceutical and nutraceutical applications owing to its high antioxidant activity. Biotechnological approaches such as genetic transformation methods (Agrobacterium-mediated) and cloning strategies, are essential to improve/regulate this ketocarotenoid in Haematococcus. For this studies are necessary to improve Haematococcus through biotechnological means. In this connection, a suitable cocultivation medium for Haematococcus and Agrobacterium tumefaciens was standardized and different antibiotics were screened. Among the cocultivation media viz Z₈, Bold’s Basal Medium, Tris Acetate Phosphate Z₈ with 0.5% mannitol and BBM and Z₈ with half strength LB TAP medium was found suitable for growth of both the alga Haematococcus and Agrobacterium. For the different antibiotics screened to identify the sensitivity of algae, hygromycin at more than 2 mg L⁻¹ showed lethal effect which can be used as a selectable marker gene in genetic transformation, whereas cefotaxime and augmentin showed no effect up to 2,000 mg L⁻¹. The growth of algae was higher in solid selection medium when compared to the liquid selection medium.     

Zerumbone induced apoptosis in liver cancer cells via modulation of Bax/Bcl-2 ratio

Background  

Zerumbone is a cytotoxic component isolated from Zingiber zerumbet Smith, a herbal plant which is also known as lempoyang. This new anticancer bioactive compound from Z. zerumbet was investigated for its activity and mechanism in human liver cancer cell lines.     

Rhamnolipids: diversity of structures, microbial origins and roles

Rhamnolipids are glycolipidic biosurfactants produced by various bacterial species. They were initially found as exoproducts of the opportunistic pathogen Pseudomonas aeruginosa and described as a mixture of four congeners: α-L-rhamnopyranosyl-α-L-rhamnopyranosyl-β-hydroxydecanoyl-β-hydroxydecanoate (Rha-Rha-C₁₀-C₁₀), α-L-rhamnopyranosyl-α-L-rhamnopyranosyl-β-hydroxydecanoate (Rha-Rha-C₁₀), as well as their mono-rhamnolipid congeners Rha-C₁₀-C₁₀ and Rha-C₁₀. The development of more sensitive analytical techniques has lead to the further discovery of a wide diversity of rhamnolipid congeners and homologues (about 60) that are produced at different concentrations by various Pseudomonas species and by bacteria belonging to other families, classes, or even phyla. For example, various Burkholderia species have been shown to produce rhamnolipids that have longer alkyl chains than those produced by P. aeruginosa. In P. aeruginosa, three genes, carried on two distinct operons, code for the enzymes responsible for the final steps of rhamnolipid synthesis: one operon carries the rhlAB genes and the other rhlC. Genes highly similar to rhlA, rhlB, and rhlC have also been found in various Burkholderia species but grouped within one putative operon, and they have been shown to be required for rhamnolipid production as well. The exact physiological function of these secondary metabolites is still unclear. Most identified activities are derived from the surface activity, wetting ability, detergency, and other amphipathic-related properties of these molecules. Indeed, rhamnolipids promote the uptake and biodegradation of poorly soluble substrates, act as immune modulators and virulence factors, have antimicrobial activities, and are involved in surface motility and in bacterial biofilm development.     

Antimicrobial Browning-Inhibitory Effect of Flavor Compounds in Seaweeds

Since ancient times, the antimicrobial properties of seaweeds have been recognized. However, antimicrobial activities of volatile compounds in seaweeds have not been explored so far. Here, essential oils from seaweeds including green, brown and red algae such as Laminaria japonica, Kjellmaniella crassifolia, Gracilaria verrucosa and Ulva pertusa were prepared by using SDE (simultaneous distillation and extraction) apparatus. Volatile compounds in the essential oils were identified as aldehydes, ketones, carboxylic acids, alcohols and hydrocarbons by comparison of GC-retention times and MS data with those of authentic specimens. Flavor compounds such as (3Z)-hexenal, (2E)-hexenal and (2E)-nonenal in some essential oils showed strong antimicrobial activities against Escherichia coli TG-1, and Erwinia carotovora. Inhibition of browning can be achieved during either of two stages, namely, oxidation reaction by tyrosinase or subsequent non-enzymatic polymerization. Tyrosinase activity was measured by monitoring absorbance at 475 nm originating from dopachrome formed from L-DOPA. Many kinds of aliphatic carboxylic acids, aldehydes and alcohols were used as inhibitors for PPO activity. The results indicated that the α,β-unsaturated carbonyl compounds strongly inhibit tyrosinase activity. When seaweeds are damaged or macerated, the α,β-unsaturated aldehydes such as (2E)-hexenal and (2E)-nonenal are biosynthesized via the corresponding (3Z)-unsaturated aldehydes from linolenic acid and arachidonic acid. The flavor compounds that are formed could be valuable as safe antimicrobial browning-inhibitory agents of edible seaweed origin.     

Purification by silica gel chromatography using dialysis tubing and characterization of sophorolipids produced from <Emphasis Type="Italic">Candida bombicola</Emphasis> grown on glucose and arachidonic acid

The yeast Candida bombicola (ATCC 22214) grown on primary carbon source glucose (100 g l⁻¹) and secondary carbon, arachidonic acid (2 g l⁻¹) produced mixture of sophorolipids up to 1.44 g l⁻¹. The crude product was a heterogeneous mixture of sophorolipids, which are glycolipids of sophorose linked to the fatty acid through glycosidic bond between ω and ω−1 carbon of arachidonic acid. The derived sophorolipids were isolated by silica gel chromatography using dialysis tubing. The purified sophorolipids were characterized by ESI-MS and FT-IR. Acid hydrolysis of the resolved sophorolipids were characterized by ESI-MS for the presence of 20-hydroxy-5Z,8Z,11Z,14Z-eicosatetraenoic acid (20-HETE) and 19-hydroxy-5Z,8Z,11Z,14Z-eicosatetraenoic acid (19-HETE), compounds of pronounced pharmaceutical importance.     

The estimation of a bivariate fitness function from two samples taken from a population

Summary The fitness of animals subjected to natural selection can be defined as the probability of surviving selection for a given interval of time, or some convenient multiple of this. If the fitness is related to some measurable variablesX, Y, Z,… then the relationship is expressed mathematically in the fitness functionw(x, y, z,…) and this function can be estimated by comparing the joint distribution ofX, Y, Z,… in samples taken before and after selection. In an earlier paper (Manly, 1975) the problems involved in estimating a fitness function of one variable were discussed. In the present paper various methods for estimating a bivariate fitness function are proposed and compared on some semiartificial sample data. It is concluded that either a generalized version ofO’Donald’s (1968) method of moments or a weighted multiple regression method will be most satisfactory. Alternative methods involving assumptions of normality will need to be used with great care.     

Eight newSalmonella types

Summary Eight newSalmonella types are described.S. houten, 43; Z₄, Z₂₃ :−, was isolated from a frog,S.tuindorp, 43: Z₄, Z₃₂: −,S. vleuten, 44: fg: −,S.zeist, 18: Z₁₀: Z₆, andS.soesterberg. 21: Z₄, Z₂₃: −, were isolated from lizards. S. kralingen (8), 20: y: Z₆, was isolated from peanuts andS. assen, 21: a: −, was isolated from a swine. All these strains were isolated in the Netherlands. S.mim, 13, 22: a: 1,6, was isolated from a rat in Ghana.     

Fine-scale resolution of closely spaced pheromone and antagonist filaments by flying male Helicoverpa zea

The limits of a male moth's ability to resolve closely spaced odor filaments have been investigated. Male Helicoverpa zea normally respond to their conspecific sex pheromone blend by exhibiting an upwind flight, which culminates in source contact by at least 50% of the bioassayed individuals. When loaded onto the same filter paper source containing this hitherto attractive pheromone blend, or onto a separate filter paper and co-emitted from the same pipette source with pheromone, (Z)-11-hexadecenyl acetate severely reduced upwind flight and source contact by male H. zea. A similar level of upwind flight inhibition was recorded when the antagonist (Z)-11-hexadecenyl acetate was emitted from its own point source placed 1 mm upwind of the pheromone point source, both plumes being simultaneously emitted in a continuous mode to form a confluent strand. However, (Z)-11-hexadecenyl acetate was less effective in reducing upwind flight and source contact when it was isolated and pulsed from its own source, placed 1 mm either upwind, downwind or cross-wind of a pipette source from which pheromone was simultaneously being pulsed, such that both filaments were separated in time by 0.001–0. 003 s. These results suggest that male H. zea are able to distinguish between odor sources separated by as little as 1 mm in space and 0.001 s in time. Accepted: 31 March 1999     

(13Z)- and (9Z)-lycopene isomers are major intermediates in the oxidative degradation of lycopene by cigarette smoke and Sin-1

Abstract

The breakdown of lycopene in the presence of reactive oxygen and reactive nitrogen species has been studied in order to identify key in vitro intermediates. These compounds may in turn be produced as metabolites in the body and may have significant physiological properties, such as increased antioxidant capacity. We have studied the in vitro degradation of lycopene in solvent, in plasma and in low density lipoprotein, when challenged with freshly generated gaseous cigarette smoke or free radicals generated in situ by S-morpholinosydonimine at 37°C. The emphasis has been to establish the major intermediates and to compare the data with previous studies using different reactants. We have found that (13Z)-lycopene is the major intermediate in both cigarette smoke and S-morpholinosydonimine reactions (representing ≥60% of the converted (all-E)-lycopene at ～50% depletion). Additionally, (9Z)-lycopene and various (all-E) and (Z)-lycopene epoxides were predominant. Notably, (5Z)-lycopene appeared to be the most stable form of lycopene under the stated conditions. Previous theoretical studies of isomer thermodynamics and rotational energy barriers for carbon double bonds fully support the pattern of isomer production and stability. In contrast to β-carotene studies, nitro-derivatives of lycopene could not be detected. In conclusion, (Z)-lycopene production and (5Z)-lycopene stability may help explain elevated (Z)-lycopene in plasma over (Z)-lycopene content in lycopene-containing foods in the diet.