长春花内生细菌多样性与柑橘黄龙病菌的相关性

【目的】分析感柑橘黄龙病长春花植株与健康长春花植株不同部位内生细菌菌群结构变化,为柑橘黄龙病菌与长春花内生细菌的相关性研究提供理论基础。【方法】本研究利用兼性厌氧可培养技术、16S rDNA限制性片段长度多态性分析(Restriction fragment length polymorphism,RFLP)以及16S rDNA序列分析相结合的方法。【结果】分别从感病和健康长春花叶、茎、根的组织中分离获得67株内生细菌,与GenBank中29种细菌的相似性达到97%-100%。其中短小杆菌属(Curtobacterium sp.)、欧文氏菌属(Erwinia sp.)、蜡样芽胞杆菌(Bacillus cereus)为感病长春花内生细菌的优势菌群,鞘胺醇单胞菌属(Brevundimonas sp.)、芽胞杆菌属(Bacillus sp.)为健康长春花内生细菌的优势菌群;马胃葡萄球菌(Staphylococcus equorum)为两者的共同优势菌群。通过RFLP方法分析,感病株得到16个、健株得到23个操作分类单元(Operational TaxonomicUnits,OTUs),感病植株中除柑橘黄龙病菌Candidatus Liberibacter asiaticus外,还有丰富的CandidatusLiberibacter sp.存在。【结论】感病与健康长春花植株中均含有丰富的内生细菌,黄龙病菌的存在改变了长春花原有内生细菌的菌群结构,且菌群多样性下降。可见长春花内生细菌在一定程度上受到柑橘黄龙病菌的抑制。     

Development of a microbial community of bacterial and yeast antagonists to control wound-invading postharvest pathogens of fruits.

Two antagonists, the bacterium Pseudomonas syringae and the pink yeast Sporobolomyces roseus, against blue mold (caused by Penicillium expansum) on apple controlled this disease more effectively when combined at approximately equal biomass (50:50 of the same turbidity) than in individual applications. Addition of L-asparagine enhanced the biocontrol effectiveness of P. syringae but decreased that of S. roseus and had no significant effect when the antagonists were combined. Populations of both antagonists increased in apple wounds and were further stimulated by the addition of L-asparagine. The carrying capacity of wounds for P. syringae was not affected by S. roseus. Populations of P. syringae in wounds inoculated individually or in a 50:50 mixture with S. roseus reached the same level after 3 days at 22 degrees C. However, populations of S. roseus recovered after applications of the mixture were consistently lower than those recovered after individual applications. Similar effects were observed in in vitro tests in which populations of S. roseus grown in mixtures with P. syringae were consistently lower than those grown alone, while the populations of P. syringae were not affected by the presence of S. roseus. A total of 36 carbon and 35 nitrogen compounds were tested for utilization by both antagonists. Fourteen nitrogenous compounds were utilized by both P. syringae and S. roseus, and an additional nine compounds were utilized by P. syringae. S. roseus and P. syringae utilized 17 and 13 carbon sources, respectively; 9 sources were common to both antagonists. Populations of these antagonists in apple wounds appear to form a relatively stable community dominated by P. syringae.(ABSTRACT TRUNCATED AT 250 WORDS)     

Evaluation of endophytic colonization of Citrus sinensis and Catharanthus roseus seedlings by endophytic bacteria

Over the last few years, the endophytic bacterial community associated with citrus has been studied as an important component interacting with Xylella fastidiosa, the causal agent of citrus variegated chlorosis (CVC). This bacterium may also colonize some model plants, such as Catharanthus roseus and Nicotiana clevelandii. In the present study, we compared the endophytic colonization of Citrus sinensis and Catharanthus roseus using the endophytic bacteria Klebsiella pneumoniae. We chose an appropriate strain, K. pneumoniae 342 (Kp342), labeled with the GFP gene. This strain was inoculated onto seedlings of C. sinensis and C. roseus. The isolation frequency was determined one week after the inoculation and the endophytic colonization of K. pneumoniae was observed using fluorescence microscopy. Although the endophytic bacterium was more frequently isolated from C. roseus than from C. sinensis, the colonization profiles for both host plants were similar, suggesting that C. roseus could be used as a model plant to study the interaction between endophytic bacteria and X. fastidiosa.     

刈割对栽培长春花(Catharanthus roseus)生活史型转变的影响

为了提高栽培长春花（Catharanthus roseus）的生物碱产量,应用植物生活史型的理论和方法,研究了刈割对栽培长春花生活史型转变及其生物碱代谢的影响。运用主成分分析法（Principal Component Analysis,PCA）对刈割后的长春花后生活史型变化进行定量和定性划分,发现在对照栽培环境下生长的长春花处于DE生境,定性划分结果为SV生活史型,定量划分结果为V0.3638,S0.6174C0.0187,属于SV型。刈割使长春花的生活史型转变为V0．2847S0.6544C0.0469,属于SC型。同时,对两种生活史型的长春花中长春碱及其前体文朵灵和长春质碱的含量进行了检测分析,发现刈割后的SC型长春花不同叶位叶片中的生物碱含量均显著提高（P〈O．05）,可以为提高栽培长春花生物碱含量提供科学指导,也进一步验证了生活史型理论。     

Effect of nitric oxide on catharanthine production and growth of Catharanthus roseus suspension cells

Sodium nitroprusside (SNP) was used as the donor of nitric oxide (NO) to investigate its effect on catharanthine synthesis and the growth of Catharanthus roseus suspension cells. The results showed that SNP at high concentrations (10.0 and 20.0 mmol/L) stimulated catharanthine formation of C. roseus cells, but inhibited growth of the cells. Low concentrations of SNP (0.1 and 0.5 mmol/L) enhanced the growth of C. roseus cells, but had no effect on catharanthine synthesis. The maximum total catharanthine production was achieved by the addition of 0.5 and 10.0 mmol/L SNP to the cultures at day 0 and day 10, respectively, being about threefold of the control. NO-induced catharanthine production of C. roseus cells was strongly suppressed by jasmonic acid (JA) biosynthesis inhibitor ibuprofen (IBU) and nordihydroguaiaretic (NDGA). The result suggests that the stimulatory role of NO on catharanthine production is partially JA-dependent.     

Molecular cloning and characterisation of two calmodulin isoforms of the Madagascar periwinkle Catharanthus roseus

Involvement of Ca(2+) signalling in regulation of the biosynthesis of monoterpene indole alkaloids (MIA) in Catharanthus roseus has been extensively studied in recent years, albeit no protein of this signalling pathway has been isolated. Using a PCR strategy, two C. roseus cDNAs encoding distinct calmodulin (CAM) isoforms were cloned and named CAM1 and CAM2. The deduced 149 amino acid sequences possess four Ca(2+) binding domains and exhibit a close identity with Arabidopsis CAM isoforms (>91%). The ability of CAM1 and CAM2 to bind Ca(2+) was demonstrated following expression of the corresponding recombinant proteins. Furthermore, transient expression of CAM1-GFP and CAM2-GFP in C. roseus cells showed a typical nucleo-cytoplasm localisation of both CAMs, in agreement with the wide distribution of CAM target proteins. Using RNA blot analysis, we showed that CAM1 and CAM2 genes had a broad pattern of expression in C. roseus organs and are constitutively expressed during a C. roseus cell culture cycle, with a slight inhibitory effect of auxin for CAM1. Using RNA in situ hybridisation, we also detected CAM1 and CAM2 mRNA in the vascular bundle region of young seedling cotyledons. Finally, using specific inhibitors, we also showed that CAMs are required for MIA biosynthesis in C. roseus cells by acting on regulation of expression of genes encoding enzymes that catalyse early steps of MIA biosynthesis, such as 1-deoxy-d-xylulose 5-phosphate reductoisomerase and geraniol 10-hydroxylase.     

长春花(Catharanthus roseus)中吲哚类生物碱含量的比较

建立了反相高效液相法测定长春花中吲哚类生物碱文多灵、长春质碱和阿玛碱含量的方法,色谱柱为HiQ sil C18色谱柱（250 mm×4.6 mm,5 μm）;流动相为1%二乙胺水溶液(磷酸调pH=7.2)—甲醇—乙腈/2:1:1(V/V);流速为1 mL·min^-1;检测波长为215 nm;柱温为40℃。并采用此方法对长春花根、茎、叶、花和种子以及不同产地的长春花中的这3种生物碱进行了检测,结果表明文多灵和长春质碱主要存在于在根、茎、叶、花中,阿玛碱主要存在于种子中;并且随着地理位置的北移,长春花中的文多灵和长春质碱的含量逐渐降低,温室中人为控制长春花中的栽培条件能提高二者的含量。     

High-pressure adaptation by salt stress in a moderately halophilic bacterium obtained from open seawater

High-pressure adaptation was examined using a moderately halophilic bacterium (Micrococcus roseus), which was isolated from open seawater and capable of growing in 15% w/v NaCl (optimum NaCl concentration: 3% w/v). After treatment at 207 MPa, colony-forming units (CFUs) significantly decreased; however, the loss of integral cells after pressure was only 30% when direct cell count was performed microscopically. In order to investigate the piezotolerance of M. roseus under high pressure without morphological change, the survival of cells was examined under pressure at 138 MPa for 2 h. M. roseus in 3% NaCl was still sensitive to pressure at 138 MPa. However, the cells in the third generations showed remarkably increased pressure resistance, and no significant loss of viability was confirmed. Furthermore, when M. roseus was cultured in 1, 3, 5, 10 and 15% NaCl, the survival ratio proportionally increased at increased NaCl concentration. M. roseus cultured in 15% NaCl was remarkably resistant (94.7% viability) to pressure at 138 MPa, even when suspended in lower concentration of NaCl. This suggests that NaCl concentrations in growth culture affect the piezotolerance of M. roseus and that this species has an ability to adapt to high pressure.     

Development of efficient catharanthus roseus regeneration and transformation system using agrobacterium tumefaciens and hypocotyls as explants

ABSTRACT: BACKGROUND: As a valuable medicinal plant, Madagascar periwinkle (Catharanthus roseus) produces many terpenoid indole alkaloids (TIAs), such as vindoline, ajamlicine, serpentine, catharanthine, vinblastine and vincristine et al. Some of them are important components of drugs treating cancer and hypertension. However, the yields of these TIAs are low in wild-type plants, and the total chemical synthesis is impractical in large scale due to high-cost and their complicated structures. The recent development of metabolic engineering strategy offers a promising solution. In order to improve the production of TIAs in C. roseus the establishment of an efficient genetic transformation method is required. RESULTS: To develop a genetic transformation method for C. roseus, A. tumefaciens strain EHA105 was employed which harbors a binary vector pCAMBIA2301 containing a report beta-glucuronidase (GUS) gene and a selectable marker neomycin phosphotransferase II gene (NTPII). The influential factors were investigated systematically and the optimal transformation condition was achieved using hypocotyls as explants, including the sonication treatment of 10 min with 80 W, A. tumefaciens infection of 30 min and co-cultivation of 2 d in 1/2 MS medium containing 100 muM acetosyringone. With a series of selection in callus, shoot and root inducing kanamycin-containing resistance mediums, we successfully obtained stable transgenic regeneration plants. The expression of GUS gene was confirmed by histochemistry, polymerase chain reaction, and genomic southern blot analysis. To prove the efficiency of the established genetic transformation system, the rate-limiting gene in TIAs biosynthetic pathway, DAT, which encodes deacetylvindoline-4-O-acetyltransferase, was transferred into C. roseus using this established system and 9 independent transgenic plants were obtained. The results of metabolite analysis using high performance liquid chromatography (HPLC) showed that overexpression of DAT increased the yield of vindoline in transgenic plants. CONCLUSIONS: In the present study, we report an efficient Agrobacterium-mediated transformation system for C. roseus plants with 11.11 % of transformation frequency. To our knowledge, this is the first report on the establishment of A. tumefaciens mediated transformation and regeneration of C. roseus. More importantly, the C. roseus transformation system developed in this work was confirmed in the successful transformation of C. roseus using a key gene involved in TIAs biosynthetic pathway resulting in the higher accumulation of vindoline in transgenic plants.     

Substrate specificity and diastereoselectivity of strictosidine glucosidase, a key enzyme in monoterpene indole alkaloid biosynthesis

Strictosidine glucosidase (SGD) from Catharanthus roseus catalyzes the deglycosylation of strictosidine, an intermediate from which thousands of monoterpene indole alkaloids are derived. The steady-state kinetics of SGD with a variety of strictosidine analogs revealed the substrate preferences of this enzyme at two key positions of the strictosidine substrate. Additionally, SGD from C. roseus turns over both strictosidine and its stereoisomer vincoside, indicating that although this enzyme prefers the naturally occurring diastereomer, the enzyme is not completely diastereoselective. The implications of the substrate specificity of SGD in metabolic engineering efforts of C. roseus are highlighted.     

Biotransformation of (+)- and (-)-camphorquinones by plant cultured cells

Biotransformation of (+)- and (-)-camphorquinones with suspension plant cultured cells of Nicotiana tabacum and Catharanthus roseus was investigated. It was found that the plant cultured cells of N. tabacum and C. roseus reduce stereoselectively the carbonyl group of (+)- and (-)-camphorquinones to the corresponding alpha-keto alcohols.     

柑橘黄龙病寄主长春花内生细菌的分离及功能鉴定

【目的】感柑橘黄龙病长春花植株与健康长春花植株不同部位内生细菌菌群结构及功能对柑橘黄龙病菌与长春花内生细菌的相关性研究提供理论基础。【方法】利用兼性厌氧可培养技术以及植物内生菌功能特性分析相结合的方法。【结果】分别从感病和健康长春花叶、茎、根的组织中分离获得67株内生细菌,与GenBank中29种细菌的相似性达到97%-100%。其中短小杆菌属(Curtobacterium sp.)、欧文氏菌属(Erwinia sp.)、蜡样芽胞杆菌(Bacillus cereus)为感病长春花内生细菌的优势菌群,鞘胺醇单胞菌属(Brevundimonas sp.)、芽胞杆菌属(Bacillus sp.)为健康长春花内生细菌的优势菌群;马胃葡萄球菌(Staphylococcus equorum)为两者的共同优势菌群。29种内生细菌进行功能分析,其中6株内生细菌至少具有4种功能特性,分属于马胃葡萄球菌、苏云金芽孢杆菌、巨大芽孢杆菌、短小杆菌属、摩氏摩根菌(Morganella morganii)及溶杆菌属(Lysobacter sp.)5个属。【结论】感病与健康长春花植株中均含有丰富的内生细菌且差异较大,黄龙病菌的存在改变了长春花原有内生细菌的菌群结构。通过分析菌群的差异,有望找到与柑橘黄龙病菌生长相关的菌种。     

Transcript profiling of terpenoid indole alkaloid pathway genes and regulators reveals strong expression of repressors in <Emphasis Type="Italic">Catharanthus roseus</Emphasis> cell cultures

The understanding of the complexities and molecular events regulating genes and the activators involved in terpenoid indole alkaloid (TIA) metabolism is known to a certain extent in cell cultures of an important TIA yielding plant, Catharanthus roseus, though it is not yet complete. Recently, the repressors of early TIA pathway genes have also been identified. However, their roles in the regulation of TIA pathway in C. roseus cell cultures remains yet unknown. We have made a comparative profiling of genes catalyzing the important steps of 2-C methyl-D-erythritol-4-phosphate (MEP), shikimate and TIA biosynthetic pathways, their activator and repressors using macroarray, semiquantitative RT-PCR and northern analyses in a rotation culture system of C. roseus comprising differentiated and proliferated cells. Our results demonstrate that TIA biosynthetic pathway genes and their activators show variable expression pattern, which was correlated with the changes in the cellular conditions in these systems. Under similar conditions, TIA pathway repressors show strong and consistent expression. The role of repressors in the complex regulation of the TIA pathway in C. roseus cell cultures is discussed. The results were supported by HPLC data, which demonstrated that the molecular program of cellular differentiation is intimately linked with TIA pathway gene expression and TIA production in C. roseus cell cultures.     

Geraniol 10-hydroxylase, a cytochrome P450 enzyme involved in terpenoid indole alkaloid biosynthesis.

Geraniol 10-hydroxylase (G10H) is a cytochrome P450 monooxygenase involved in the biosynthesis of iridoid monoterpenoids and several classes of monoterpenoid alkaloids found in a diverse range of plant species. Catharanthus roseus (Madagascar periwinkle) contains monoterpenoid indole alkaloids, several of which are pharmaceutically important. Vinblastine and vincristine, for example, find widespread use as anti-cancer drugs. G10H is thought to play a key regulatory role in terpenoid indole alkaloid biosynthesis. We purified G10H from C. roseus cells. Using degenerate PCR primers based on amino acid sequence information we cloned the corresponding cDNA. The encoded CYP76B6 protein has G10H activity when expressed in C. roseus and yeast cells. The stress hormone methyljasmonate strongly induced G10h gene expression coordinately with other terpenoid indole alkaloid biosynthesis genes in a C. roseus cell culture.     

Metabolic discrimination of Catharanthus roseus leaves infected by phytoplasma using 1H-NMR spectroscopy and multivariate data analysis

A comprehensive metabolomic profiling of Catharanthus roseus L. G. Don infected by 10 types of phytoplasmas was carried out using one-dimensional and two-dimensional NMR spectroscopy followed by principal component analysis (PCA), an unsupervised clustering method requiring no knowledge of the data set and used to reduce the dimensionality of multivariate data while preserving most of the variance within it. With a combination of these techniques, we were able to identify those metabolites that were present in different levels in phytoplasma-infected C. roseus leaves than in healthy ones. The infection by phytoplasma in C. roseus leaves causes an increase of metabolites related to the biosynthetic pathways of phenylpropanoids or terpenoid indole alkaloids: chlorogenic acid, loganic acid, secologanin, and vindoline. Furthermore, higher abundance of Glc, Glu, polyphenols, succinic acid, and Suc were detected in the phytoplasma-infected leaves. The PCA of the (1)H-NMR signals of healthy and phytoplasma-infected C. roseus leaves shows that these metabolites are major discriminating factors to characterize the phytoplasma-infected C. roseus leaves from healthy ones. Based on the NMR and PCA analysis, it might be suggested that the biosynthetic pathway of terpenoid indole alkaloids, together with that of phenylpropanoids, is stimulated by the infection of phytoplasma.     

Efficacy of foliar extracts against pre- and post-harvest diseases of sponge-gourd fruits

S.K. AHMAD AND J.S. PRASAD. 1995. The inhibitory potential of aqueous foliar extracts of Adhatoda vasica, Azadirachta indica, Catharanthes roseus, Datura fistulosa, Lantana camara, Muraya exotica, Ocimum sanctum, Ricinus communis and Strychnos nux-vomica were evaluated against soft rot diseases of sponge-gourd fruits caused by Helminthosporium spiciferum and Fusarium scirpi. Conidial germination of F. scirpi and H. spiciferum were reduced to about 75% when their spores were treated with A. indica, C. roseus, D. fistulosa, L. camara, M. exotica and O. sanctum. Considerable reductions (50%) in mycelial dry weights and colony diameters were recorded in liquid media containing (20%) aqueous extracts of C. roseus, D. fistulosa and M. exotica . Other extracts reduced the mycelial growth but to a lesser extent. Post-infection treatment of sponge-gourd fruits with the extracts of A. indica, L. camara, M. exotica, O. sanctum, D. fistulosa and C. roseus almost fully inhibited the spread of disease. Treatment of fruits with A. indica and C. roseus before fungal infection reduced the spread of disease caused by both the pathogens, whereas M. exotica, O. sanctum and D. fistulosa reduced the spread of soft-rot caused by F. scirpi but not by H. spiciferum .