Integrated management of bacterial wilt (Ralstonia solanacearum) of ginger (Zingiber officinale) in Southwestern Ethiopia

Abstract

Ginger bacterial wilt (GBW) is a destructive disease of ginger in Ethiopia. Field studies were conducted to determine effect of integrated management of GBW, through host resistance and cultural practices, on wilt epidemics at Teppi and Jimma, southwestern Ethiopia in 2017. Treatments were factorial arranged in a randomised complete block design with three replications. Analysis of variance indicated that effect of variety, cultural practices and variety x cultural practice interactions significantly reduced GBW epidemic parameters and enhanced yield at both locations. Boziab recorded lower wilt incidence, area under disease progress curve and wilt progress rates than Local variety. Integration of lemon grass with soil solarisation and fertiliser reduced wilt incidence in Local variety up to 38.3% (Teppi) and 42.05% (Jimma) compared to the control on final wilt assessment date. In Boziab variety, integrated use of lemon grass with soil solarisation and fertiliser reduced wilt incidence up to 42.5% at Teppi and 33.85% at Jimma compared to the control on final date of wilt assessment. The overall results revealed that integrating cultural practices with host resistance are found effective to slow down GBW epidemics and improve ginger productivity; and thus, recommended for the study areas along with other crop management practices.     

Cloning, expression, purification and characterization of recombinant (+)-germacrene D synthase from Zingiber officinale

A cDNA clone encoding a sesquiterpene synthase, (+)-germacrene D synthase, has been isolated from ginger (Zingiber officinale). The full-length cDNA (AY860846) contains a 1650-bp open reading frame coding for 550 amino acids (63.8kDa) with a theoretical pI=5.59. The deduced amino acid sequence is 30-46% identical with sequences of other sesquiterpene synthases from angiosperms. The recombinant enzyme, produced in Escherichia coli, catalyzed the formation of a major product, (+)-germacrene D (50.2% of total sesquiterpenoids produced) and a co-product, germacrene B (17.1%) and a number of minor by-products. The optimal pH for the recombinant enzyme is around 7.5. Substantial (+)-germacrene D synthase activity is observed in the presence of Mg2+, Mn2+, Ni2+ or Co2+, while the enzyme is inactive when Cu2+ or Zn2+ is used. The Km- and kcat-values are 0.88 microM and 3.34 x 10(-3) s(-1), respectively. A reaction mechanism involving a double 1,2-hydride shift has been established using deuterium labeled substrates in combination with GC-MS analysis.     

In vitro effectiveness of Curcuma longa and Zingiber officinale extracts on Echinococcus protoscoleces

Hydatid disease is an important economic and human public health problem with a wide geographical distribution. Surgical excision remains the primary treatment and the only hope for complete cure of hydatosis. The most important complications arising from surgical excision, however, is recurrence, which is due to dissemination of protoscolices during the surgery. Pre-surgical inactivation of the contents of the hydatid cyst by injection of scolicidal agent into the cyst has been used as adjunct to surgery in order to overcome the risk of recurrence. In the present study, ethanolic extracts of turmeric (Curcuma longa) and ginger (Zingiber officinale) were tested as scolicidal agent for Echinococcus protoscoleces. Protoscoleces were collected aseptically from sheep livers containing hydatid cysts. Three concentrations (10, 30 and 50 mg/ml) of each extract were investigated and viability of the protoscoleces was tested by 0.1% eosin staining. Ginger extract showed the strongest scolicidal effect (100%) after 20 min at a concentration of 30 mg/ml and 10 min at 50 mg/ml. The maximum scolicidal effect of turmeric was 93.2% after 30 min at a concentration of 50 mg/ml. It is concluded that turmeric and ginger extracts have high scolicidal activity and could be used as effective scolicidal agents against Echinococcus protoscoleces.     

抗青枯病基因RRS1的研究进展

RRS1是被发现的第一个青枯病抗性基因,能介导多个Ralstonia solanacearum小种的广谱抗性反应,也是至今第一例依赖NDR1蛋白的TIR-NBS-LRR类R基因。该文综述了目前分子机制研究最为详尽的拟南芥抗青枯病基因RRS1的克隆、功能和表达作用模式的研究进展,对其他作物青枯病抗性的分子机理研究提供科学的依据和启示。     

Isolation and characterization of antioxidant and antibacterial compound from mango ginger (Curcuma amada Roxb.) rhizome

The chloroform extract of mango ginger (Curcuma amada Roxb.) rhizome was subjected to antioxidant activity-guided purification by repeated silica gel column chromatography to obtain a pure antioxidant compound. The structure was deduced by analyzing UV, IR, liquid chromatography-mass spectrometry (LC-MS) and two-dimensional heteronuclear multiple quantum coherence transfer spectroscopy (2D-HMQCT) NMR spectral data, and named it as "Amadannulen", a novel compound. It exhibited DPPH radical scavenging activity, super oxide radical scavenging activity, lipid peroxidation inhibitory activity and metal chelating activity. Amadannulen also showed antibacterial activity against both Gram-positive and Gram-negative bacteria tested. It also exhibited bactericidal activity against M. luteus, B. cereus and B. subtilis.     

In vitro clonal multiplication of turmeric (Curcuma spp.) and ginger (Zingiber officinale Rosc.)

Rhizome buds, excised from threeCurcuma spp., and ginger, inoculated aseptically on MS medium with varying levels of BAP and kinetin, produced multiple shoots. For shoot multiplication, a concentration of 3.0 mg/l BAP was found to be optimum for all the species.In vitro plants were successfully established in the field and were morphologically uniform. A simple method to extend the subculture interval was used and its relevance to germplasm conservation is discussed.Abbreviations BAP 6-benzylaminopurine - kinetin 6-furfurylaminopurine - MS Murashige and Skoog (1962)     

Genetic mapping of a major dominant gene for resistance to Ralstonia solanacearum in eggplant

Resistance of eggplant against Ralstonia solanacearum phylotype I strains was assessed in a F₆ population of recombinant inbred lines (RILs) derived from a intra-specific cross between S. melongena MM738 (susceptible) and AG91-25 (resistant). Resistance traits were determined as disease score, percentage of wilted plants, and stem-based bacterial colonization index, as assessed in greenhouse experiments conducted in Réunion Island, France. The AG91-25 resistance was highly efficient toward strains CMR134, PSS366 and GMI1000, but only partial toward the highly virulent strain PSS4. The partial resistance found against PSS4 was overcome under high inoculation pressure, with heritability estimates from 0.28 to 0.53, depending on the traits and season. A genetic map was built with 119 AFLP, SSR and SRAP markers positioned on 18 linkage groups (LG), for a total length of 884 cM, and used for quantitative trait loci (QTL) analysis. A major dominant gene, named ERs1, controlled the resistance to strains CMR134, PSS366, and GMI1000. Against strain PSS4, this gene was not detected, but a significant QTL involved in delay of disease progress was detected on another LG. The possible use of the major resistance gene ERs1 in marker-assisted selection and the prospects offered for academic studies of a possible gene for gene system controlling resistance to bacterial wilt in solanaceous plants are discussed.     

温郁金萜类合酶基因CwTPS4的克隆和表达特征

温郁金是著名的"浙八味"之一,药用价值高、应用广泛,萜类化合物是其主要的药用成分.萜类合酶是植物萜类化合物生物合成途径中的关键酶.依据温郁金根茎的转录组数据,采用反转录PCR获得了1个萜类合酶基因CwTPS4(GenBank登录号:MW774935),其开放阅读框长1515 bp,编码504个氨基酸,含有萜类合酶特有的结构域和保守序列RXR、DDXXD等;生物信息学分析表明CwTPS4编码的蛋白定位在细胞质中、无跨膜区域,为水溶性的稳定蛋白,属于萜类合酶的TPS-a亚家族成员.实时荧光定量分析表明,CwTPS4基因主要在生长旺盛的叶片中表达,其次在根茎膨大初期的根茎中表达量较高,而在成熟或衰老的组织中表达量较低.本研究从温郁金中克隆得到1个新的萜类合酶基因CwTPS4,经生物信息学分析表明CwTPS4可能参与了温郁金中倍半萜类化合物的合成,为下一步研究其在温郁金萜类物质合成中的功能奠定了一定的基础.     

Genomic structure and phylogeny of the plant pathogen Ralstonia solanacearum inferred from gene distribution analysis

In the present study, we investigated the gene distribution among strains of the highly polymorphic plant pathogenic beta-proteobacterium Ralstonia solanacearum, paying particular attention to the status of known or candidate pathogenicity genes. Based on the use of comparative genomic hybridization on a pangenomic microarray for the GMI1000 reference strain, we have defined the conditions that allowed comparison of the repertoires of genes among a collection of 18 strains that are representative of the biodiversity of the R. solanacearum species. This identified a list of 2,690 core genes present in all tested strains. As a corollary, a list of 2,338 variable genes within the R. solanacearum species has been defined. The hierarchical clustering based on the distribution of variable genes is fully consistent with the phylotype classification that was previously defined from the nucleotide sequence analysis of four genes. The presence of numerous pathogenicity-related genes in the core genome indicates that R. solanacearum is an ancestral pathogen. The results establish the long coevolution of the two replicons that constitute the bacterial genome. We also demonstrate the clustering of variable genes in genomic islands. Most genomic islands are included in regions with an alternative codon usage, suggesting that they originate from acquisition of foreign genes through lateral gene transfers. Other genomic islands correspond to genes that have the same base composition as core genes, suggesting that they either might be ancestral genes lost by deletion in certain strains or might originate from horizontal gene transfers.     

Characteristic expression of wheat PR5 gene in response to infection by the leaf rust pathogen,Puccinia triticina

TaLr35PR5 gene was obtained from the gDNA and cDNA of TcLr35 wheat. It was induced by Puccinia triticina, ABA and SA, but TaLr35PR5 was induced earlier and its expression level was higher in the incompatible interaction than that in the compatible interaction. In addition, the accumulations of TaLr35PR5 increased stably and showed significant peak challenged by P. triticina at different growth and development periods of TcLr35 wheat while it maintained similar level and changed little in mock inoculated. Western blottings were conducted to confirm that TaLr35PR5 be induced by P. triticina infection at the protein expression level. Similar to the expression patterns of TaLr35PR5 at RNA levels, the accumulations of TaLr35PR5 protein were weak in the seedling stage, then increased to the peak and kept constant levels at the mature stage which is consistent with the expression feature of Lr35 gene as an adult plant resistance gene.     

Host plant-dependent phenotypic reversion of Ralstonia solanacearum from non-pathogenic to pathogenic forms via alterations in the phcA gene

Ralstonia solanacearum is a plant pathogenic bacterium that undergoes a spontaneous phenotypic conversion (PC) from a wild-type pathogenic to a non-pathogenic form. PC is often associated with mutations in phcA, which is a key virulence regulatory gene. Until now, reversion to the wild-type pathogenic form has not been observed for PC variants and the biological significance of PC has been questioned. In this study, we characterized various alterations in phcA (eight IS element insertions, three tandem duplications, seven deletions and a base substitution) in 19 PC mutants from the model strain GMI1000. In five of these variants, reversion to the pathogenic form was observed in planta, while no reversion was ever noticed in vitro whatever culture media used. However, reversion was observed for a 64 bp tandem duplication in vitro in the presence of tomato root exudate. This is the first report showing a complete cycle of phenotypic conversion/reversion in a plant pathogenic bacterium.     

Cloning and sequencing of PR5-like gene from high altitude adapted ecotype of Lepidium latifolium

PR5-like gene (LlPR5) identified from substracted cDNA library of Lepidium latifolium in response to cold stress was characterised. Based on the EST sequence (FG618347) of a cDNA fragment, the full-length cDNA of 1422 bp was cloned by rapid amplification of cDNA ends. This LlPR5 has 931 bp of CDS encoding a protein of 326 amino acids with molecular weight of 34.46 kDa. Phylogenetic and conserved motif analysis reveals that cloned LlPR5 gene of L. latifolium formed cluster with At1g75800 of Arabidopsis thaliana. In silico promoter analysis of homolog of LlPR5 gene from Arabidopsis genome identified cis elements with possible role in regulation of cold/low temperature response in addition to its role in various stresses induced by pathogens in the plants.