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.     

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.     

Phenazine carboxylic acid production and rhizome protective effect of endophytic Pseudomonas aeruginosa isolated from Zingiber officinale

Ginger (Zingiber officinale) is cultivated commercially in most parts of the world especially in India for its culinary and medicinal applications. One of the major challenges that limit the yield of ginger is rhizome rot disease caused by organisms including Pythium myriotylum. A feasible ecofriendly method is yet to be devised to prevent the plant from this threatening disease. Recent studies on plant microbiome show the possibility of having endophytic organisms with plant protective characteristics associated with the plants. Because of the uniquely evolved underground nature of the ginger rhizome and its peculiar survival in soil for a long time, many interesting endophytic microbes with plant protective characters can be well expected from it. In the current study, previously isolated endophytic Pseudomonas aeruginosa from ginger was investigated in detail for its effect on Pythium myriotylum. The rhizome protective effect of the organism was also studied by co-inoculation studies, which confirmed that Pseudomonas aeruginosa has very potent inhibitory effect on Pythium myriotylum. On further studies, the active antifungal compound was identified as phenazine 1-carboxylic acid.     

Green synthesis of gold nanoparticles with Zingiber officinale extract: Characterization and blood compatibility

Biosynthesis of gold nanoparticles with small size and biostability is very important and used in various biomedical applications. There are lot of reports for the synthesis of gold nanoparticles by the addition of reducing agent and stabilizing agent. In the present study we have synthesized gold nanoparticles, with a particle size ranging from 5 to 15 nm, using Zingiber officinale extract which acts both as reducing and stabilizing agent. Z. officinale extract is reported to be a more potent anti-platelet agent than aspirin. Therefore, green synthesis of gold nanoparticles with Z. officinale extract, as an alternative to chemical synthesis, is beneficial from its biological and medical applications point of view, because of its good blood biocompatibility and physiological stability. The formation and size distribution of gold nanoparticles were confirmed by dynamic light scattering (DLS), UV–vis spectrophotometer and transmission electron microscopy (TEM). Gold nanoparticles synthesized using citrate and Z. officinale extract demonstrated very low protein adsorption. Both nanoparticles were non platelet activating and non complement activating on contact with whole human blood. They also did not aggregate other blood cells, however, nanoparticles synthesised with Z. officinale extract was highly stable at physiological condition compared to citrate capped nanoparticles, which aggregated. Thus the usage of nanoparticles, synthesized with Z. officinale extract, as vectors for the applications in drug delivery, gene delivery or as biosensors, where a direct contact with blood occurs is justified.     

Biological and chemical properties of Zingiber zerumbet Smith: a review

Numerous researches have been carried out in Zingiber zerumbet Smith. Since 1944 till date. Z. zerumbet is a monocotyledonous perennial medicinal plant belonging to Zingiberaceae family. It is commonly known as shampoo ginger. It has many different local names depending on their area of collection and vegetation. It is called as ‘Singkha’ in Manipuri. Various compounds have been reported to be isolated from Z. zerumbet and they serve a very potent and reliable drug candidate for the various diseases. They have been investigated for its prospects of effectiveness against number of activities in in vitro as well as in vivo and mechanisms that may be involved in chemo preventive measures and various pharmaceutical studies.     

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.     

生姜根茎的发育过程及分泌腔的超微结构

为了解生姜(Zingiber officinale Roscoe)根茎的发育过程,在光学显微镜和电子显微镜下对不同发育时期的生姜进行显微和超微结构观察,并对分泌腔的发生发育过程进行了研究。结果表明,幼嫩期的生姜,表皮以内的基本组织可大致分为皮层、拟内皮层和中柱。次生加厚分生组织起源于中柱外侧一些细胞,细胞分裂和体积增大促使生姜发育。薄壁细胞内有大量的淀粉粒且其数量、形状和大小因发育时期而不同。分泌腔广布于生姜中,其发育过程可分为3个阶段:分泌腔原始细胞团形成、分泌腔的发生和成熟分泌腔形成。生姜精油主要在线粒体、质体和细胞质中合成。本研究为生姜药用资源的开发利用提供了理论依据。     

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.     

Characterization of polymorphic microsatellite markers,isolated from ginger (Zingiber officinale Rosc.)

The present study reports isolation and characterization of eight polymorphic microsatellite markers for Zingiber officinale Rosc. (Ginger). A total of 34 alleles were detected across the 20 accessions, with an average of 4.3 alleles per locus. Values for observed and expected heterozygosities ranged from 0 to 1.0 and from 0.23 to 0.67, respectively. The heterozygote deficits were observed at three loci. At the significance threshold (P < 0.05) of the eight loci, seven were found to have deviated from Hardy–Weinberg equilibrium, whereas significant linkage disequilibria were observed between 10 pairs of loci. Our data indicate the existence of moderate level of genetic diversity among the ginger accessions genotyped with eight markers.     

High-frequency in vitro multiplication of disease-free Zingiber officinale Rosc.

High-frequency in vitro multiplication of disease-free clones of ginger (Zingiber officinale Rosc.) was obtained by culturing small and active buds of ginger on MS medium supplemented with 2 mg/l Kin and 20 g/l sucrose. An average of 7.7 shoots per bud was obtained on this medium after 4 weeks of culture. A high multiplication rate of well-developed plantlets (7.0 shoots per bud) with a 6.8-cm shoot length and a 7.0-cm root length was also obtained on MS medium containing 2.0 mg Kin, 2.0 mg NAA and 20 g sucrose per liter. The multiplication rate did not decrease even up to 28 months of subculture on the same medium. A simple method of successfully transferring more than 95% of tissue-cultured plants into pots was also standardized. In vitro-derived plants performed well under field conditions, were morphologically identical to the mother plants and were free from ginger yellows (Fusarium oxysporum f. sp. zingiberi). Well-developed rhizomes obtained from the tissue-cultured plants did not rot during storage of up to 6 months, thus indicating that the method is also effective in checking storage rot caused by F. oxysporum f. sp. zingiberi. Received: 25 September 1996 / Revision received: 7 May 1997 / Accepted: 3 June 1997     

Control of stored grain pest,Callosobruchus maculatus (F.) (Coleoptera: Bruchidae), using the essential oils isolated from Zingiber officinale (L.) and Mentha pulegium (L.) in laboratory condition

Fumigant activity of essential oil vapours distilled from Zingiber officinale (L.) and Mentha pulegium (L.) was tested against eggs, larvae and adults of Callosobruchus maculatus (F.). Fumigant toxicity was assessed at 27?±?1?°C and 60?±?5% RH, in dark condition. The influence of different concentrations of the essential oil vapours on egg hatchability, larval and adult mortality was significant. Data probit analysis showed that lethal concentration of the essential oil to kill 50% of the population (LC₅₀) for egg, larvae and adult was found to be 1.151, 2.336 and 2.183?μl/l air of Z. officinale, followed by 0.072, 0.113 and 0.093?μl/l air essential oil of M. pulegium, respectively. Between these essential oils, Z. officinale is almost more toxic than M. pulegium on all growth stages of C. maculatus. The present study suggests that essential oils from these medicinal plants may be potential grain protectants as botanical alternative fumigants and could be used in the management of various life stages of C. maculatus.     

Genetic diversity analysis of Zingiber Officinale Roscoe by RAPD collected from subcontinent of India

The present investigation was undertaken for the assessment of 12 accessions of Zingiber officinale Rosc. collected from subcontinent of India by RAPD markers. DNA was isolated using CTAB method. Thirteen out of twenty primers screened were informative and produced 275 amplification products, among which 261 products (94.90%) were found to be polymorphic. The percentage polymorphism of all 12 accessions ranged from 88.23% to 100%. Most of the RAPD markers studied showed different levels of genetic polymorphism. The data of 275 RAPD bands were used to generate Jaccard’s similarity coefficients and to construct a dendrogram by means of UPGMA. Results showed that ginger undergoes genetic variation due to a wide range of ecological conditions. This investigation was an understanding of genetic variation within the accessions. It will also provide an important input into determining resourceful management strategies and help to breeders for ginger improvement program.     

Anti-inflammatory effect of the hydralcoholic extract of Zingiber officinale rhizomes on rat paw and skin edema.

Plant extracts have been used for centuries as a popular mode of treatment for several health disorders. Over the last ten years, the study of those extracts has attracted attention in different fields of the biological sciences. Ginger, the rhizome of Zingiber officinale Roscoe (Zingiberaceae), is a commom constituent of diet worldwide and it has been reported that its extracts present some pharmacological activities. Here we investigate the effects of the crude hydralcoholic extract of ginger rhizomes on the classical models of rat paw and skin edema. The carrageenan-, compound 48/80- or serotonin-induced rat paw edema were inhibited significantly by the intraperitoneal administration of alcoholic ginger extract. Ginger extract was also effective in inhibiting 48/80-induced rat skin edema at doses of 0.6 and 1.8 mg/site. Rat skin edema induced by substance P or bradikinin was not affected by treatment with Z. officinalle extract. The intraperitoneal administration of ginger extract (186 mg/kg(-1) body wt.) 1 h prior to serotonin injections, reduced significantly the serotonin-induced rat skin edema. Our results demonstrated that crude extract of Zingiber officinale was able to reduce rat paw and skin edema induced by carrageenan, 48/80 compound and serotonin. The antiedematogenic activity seems to be related, at least partially, to an antagonism of the serotonin receptor.     

Cloning and characterization of PR5 gene from Curcuma amada and Zingiber officinale in response to Ralstonia solanacearum infection

Ginger (Zingiber officinale Roscoe), is an important spice crop that is badly affected by Ralstonia solanacearum wilt. Ginger does not set seed and sexual recombination has never been reported. In spite of extensive search in its habitats, no resistance source to Ralstonia induced bacterial wilt, could be located in ginger. Curcuma amada Roxb. is a potential donor for bacterial wilt resistance to Z. officinale, if the exact mechanism of resistance is understood. Pathogenesis-related (PR)-5 proteins are a family of proteins that are induced by different phytopathogens in many plants and share significant sequence similarity with thaumatin. Two putative PR5 genes, CaPR5 and ZoPR5, were amplified from C. amada and ginger, which encode precursor proteins of 227 and 224 amino acid residues, respectively, and share high homology with a number of other PR5 genes. The secondary and three-dimensional structure comparison did not reveal any striking differences between these two proteins. The expression of Ca and ZoPR5s under R. solanacearum inoculation was analyzed at different time points using quantitative real-time PCR (qRT-PCR). Our results reveal that CaPR5 is readily induced by the bacterium in C. amada, while ZoPR5 induction was very weak and slow in ginger. These results suggest that the CaPR5 could play a role in the molecular defense response of C. amada to pathogen attack. This is the first report of the isolation of PR5 gene from the C. amada and Z. officinale. Promoter analysis indicates the presence of a silencing element binding factor in ZoPR5-promoter, but not in CaPR5. Prospective promoter elements, such as GT-1 box and TGTCA, implicated as being positive regulatory elements for expression of PR proteins, occur in the 5′-flanking sequences of the CaPR5. Transient GUS expression study confirms its action with a weaker GUS expression in ginger, indicating that the PR5 expression may be controlled in the promoter.     

Essential Oil of Zingiber cassumunar Roxb. and Zingiber officinale Rosc.: A Comparative Study on Chemical Constituents,Antibacterial Activity,Biofilm Formation,and Inhibition of Pseudomonas aeruginosa Quorum Sensing System

Pseudomonas aeruginosa can regulate its pathogenicity via quorum sensing (QS) system. Zingiber cassumunar and Z. officinale have been used for the treatment of infectious diseases. The study aimed to evaluate and compare the chemical constituents, antibacterial, and QS inhibitor of Z. cassumunar essential oils (ZCEO) and Z. officinale essential oils (ZOEO). The chemical constituent was analysed using GC/MS. Broth microdilution and spectrophotometry analysis were used to evaluate their antibacterial and QS inhibitor activities. The main constituent of ZOEO with percent composition above 6 % (α-curcumene, α-zingiberene, β-sesquiphellandrene, and β-bisabolene, α-citral, and α-farnesene) were exist in a very minimal percentage less than 0.7 % in Z. cassumunar. All major components of ZCEO with percentages higher than 5 % (terpinen-4-ol, sabinene, γ-terpinene) were present in low proportion (<1.18 %) in Z. officinale. ZCEO demonstrated moderate antibacterial activity against P. aeruginosa. The combination of ZCEO and tetracycline showed a synergistic effect (FICI of 0.5). ZCEO exhibited strong activity in inhibiting biofilm formation. ZCEO at MIC (62.5 μg/mL) was able to reduce pyoverdine, pyocyanin, and proteolytic activity. This is the first report on the activity of ZCEO in the inhibition of P. aeruginosa QS system and it may be used to control the pathogenicity of P. aeruginosa.     

Differential Responses of Host and Non-host Substrata on Germination of Ascospores of Sclerotinia sclerotiorum

Effect of host.(Cicer arietinum L.) and some non-host (Allium cepa L., A. Sativum L., Ocimum sanictum L., Azadirachta indica Juss., Zingiber officinale Roscoe. And Curcuma longa L.) substrata on the germination of ascospores io Sclerotinia sclerotiorum (Lib.) de Bary has been observed. Maximum germination was noticed on flower petals of gram (C.arietinum) with minimum time (2.5 h) for germ tube initiation. Among the non-host substrate germination was completely inhibited on ginger rhizome peeling whereas delayed germination (after 12h) and lowest germination percentage (48%) as compared with other non-hosts, were observed on turmeric rhizome peeling. It is suggested that ginger extract may be effective in controlling stem rot and wilt of gram incited by S. sclerotiorum in the field.     

Efficient functional analysis system for cyanobacterial or plant cytochromes P450 involved in sesquiterpene biosynthesis

Tractable plasmids (pAC-Mv-based plasmids) for Escherichia coli were constructed, which carried a mevalonate-utilizing gene cluster, towards an efficient functional analysis of cytochromes P450 involved in sesquiterpene biosynthesis. They included genes coding for a series of redox partners that transfer the electrons from NAD(P)H to a P450 protein. The redox partners used were ferredoxin reductases (CamA and NsRED) and ferredoxins (CamB and NsFER), which are derived from Pseudomonas putida and cyanobacterium Nostoc sp. strain PCC 7120, respectively, as well as three higher-plant NADPH-P450 reductases, the Arabidopsis thaliana ATR2 and two corresponding enzymes derived from ginger (Zingiber officinale), named ZoRED1 and ZoRED2. We also constructed plasmids for functional analysis of two P450s, α-humulene-8-hydroxylase (CYP71BA1) from shampoo ginger (Zingiber zerumbet) and germacrene A hydroxylase (P450NS; CYP110C1) from Nostoc sp. PCC 7120, and co-transformed E. coli with each of the pAC-Mv-based plasmids. Production levels of 8-hydroxy-α-humulene with recombinant E. coli cells (for CYP71BA1) were 1.5- to 2.3-fold higher than that of a control strain without the mevalonate-pathway genes. Level of the P450NS product with the combination of NsRED and NsFER was 2.9-fold higher than that of the CamA and CamB. The predominant product of P450NS was identified as 1,2,3,5,6,7,8,8a-octahydro-6-isopropenyl-4,8a-dimethylnaphth-1-ol with NMR analyses.