溶磷细菌和丛枝菌根真菌接种对南方红豆杉生长及根际微生物和土壤酶活性的影响

以南方红豆杉实生苗为材料,采用盆栽实验探讨了高效溶磷细菌草木樨中华根瘤菌(Sinorhizobium meliloti)CHW10B与丛枝菌根真菌缩球囊霉(Glomus constrictum)单独和双接种条件下,其植株生长、根际土壤可培养微生物数量、土壤酶活和土壤微生物功能多样性变化,在微生态水平揭示接种对南方红豆杉生长的影响及其机制。结果表明:(1)各接种处理对南方红豆杉幼苗均有促生长作用,接种处理苗高、地径和生物量均较对照显著增加,并以双接种促进效果最好。(2)各接种处理提高了南方红豆杉根际土壤可培养细菌、真菌和放线菌含量,增加了土壤微生物碳源利用率,改变了土壤中物种的丰富度和均一度,增加了土壤中的生物多样性。(3)各接种处理促进了南方红豆杉根际重要土壤酶(酸性磷酸酶、脱氢酶、转化酶)活力的增加,且双接种的促进作用最为明显。可见,溶磷细菌(草木樨中华根瘤菌CHW10B)和丛枝菌根真菌(缩球囊霉)具有协同作用,两者同时接种可显著提高南方红豆杉根际土壤微生物数量及土壤酶活力,提高土壤微生物碳源利用率和土壤肥力,增加土壤中的生物多样性,从而达到间接促进宿主植物南方红豆杉生长的目的。     

南方红豆杉水浸提液对喜树种子发芽和幼苗生长的化感作用

化感作用是影响人工混交林种间关系的重要因素之一。已有研究表明：喜树南方红豆杉混交对喜树生长有明显促进作用,并从混交改善混交林地微气候角度解释了这种促进作用,但未从种间化感作用角度探讨这种作用。为了探究南方红豆杉是否对喜树具有潜在的化感促进作用,从而更全面了解喜树南方红豆杉混交林种间关系,考察了不同浓度（25、50 g/L和100 g/L）的南方红豆杉（Taxus chinensis var.mairei）鲜叶、凋落叶、枝和根水浸提液对喜树（Camptotheca acuminata）种子发芽和幼苗生长的影响。结果表明：（1）南方红豆杉凋落叶浸提液对喜树种子发芽和幼苗生长无显著影响（P>0.05）,而鲜叶、枝和根浸提液对喜树发芽和幼苗生长均具有不同程度的促进作用（P<0.05）,且作用强度大体与浓度呈正相关;（2）100 g/L的南方红豆杉鲜叶、枝和根浸提液浸泡的喜树种子,其发芽率比对照组分别提高8.1%、14.9%和25.6%;（3）南方红豆杉鲜叶浸提液只有在高浓度（100 g/L）时,对喜树幼苗基径生长具有促进作用（P<0.05）,而对其株高、全株干重和净光合速率无显著影响;南方红豆杉根和枝浸提液对喜树幼苗株高、基茎、干重和净光合速率均具有促进作用（P<0.05）,与对照组相比,100 g/L的根和枝浸提液浇灌喜树幼苗,可以使喜树幼苗株高分别提高14.2%和8.4%,基径分别提高19.0%和15.3%,干重分别提高23.1%和15.9%,净光合速率分别提高11.6%和6.1%。研究结果表明,在喜树南方红豆杉混交林中,南方红豆杉对喜树的正向化感作用可能是促进喜树生长的重要因素之一。     

Effects of <i>Piriformospora indica</i> on the Respiration of <i>Taxus chinensis</i> var. <i>mairei</i> under Water Stress

Seedlings of Taxus chinensis var. mairei were used as experimental materials to study the adaptation of Piriformospora indica to this plant under water stress. The materials were divided into two groups, namely, with or without inoculation with P. indica. Each group was subjected to four different levels of water stress. Vitality and physiological and biochemical indexes of the roots of T. chinensis var. mairei were regularly measured. Under water stress, T. chinensis var. mairei had significantly decreased root vitality; root vitality was higher in inoculated roots than in uninoculated roots. Under intense water stress, the inoculated roots had a higher soluble sugar content than the uninoculated roots. Under water stress, T. chinensis var. mairei experienced decreased activity of aerobic respiratory metabolic enzymes. The activity of anaerobic respiratory metabolic enzymes and alcohol dehydrogenase initially increased and then decreased, whereas that of lactate dehydrogenase increased. The inoculated roots had a higher activity of respiratory metabolic enzymes than the uninoculated roots. As water stress was further intensified, the roots had significantly decreased activity of aerobic respiratory metabolic enzymes and significantly increased activity of anaerobic respiratory metabolic enzymes. The activity of respiratory metabolic enzymes decreased faster in the uninoculated roots than in the inoculated roots. This study demonstrated that Piriformospora indica plays a positive role in enhancing the antihypoxic ability of T. chinensis var. mairei, thereby alleviating plant damage due to water stress.     

Paclitaxel production using co-culture of <Emphasis Type="Italic">Taxus</Emphasis> suspension cells and paclitaxel-producing endophytic fungi in a co-bioreactor

The co-culture of the suspension cells of Taxus chinensis var. mairei and its endophytic fungi, Fusarium mairei, in a 20-L co-bioreactor was successfully established for paclitaxel production. The co-bioreactor consists of two-unit tanks (10 L each) with a repairable separate membrane in the center, culturing Taxus suspension cells in one tank and growing fungi in another. By optimizing the co-culture conditions, there was a desirable yield of paclitaxel in Taxus cell cultures. The Taxus cell cultures by co-culture produced 25.63 mg/L of paclitaxel within 15 days; it was equivalent to a productivity of 1.71 mg/L per day and 38-fold higher than that by uncoupled culture (0.68 mg/L within 15 days). The optimum conditions for co-culture in the co-bioreactor were: B5 medium, inoculating fungi when Taxus cells had grown for 5 days in the co-bioreactor, hydrophilic separate membrane in the center of the co-bioreactor, and air flow rate of 1:0.85 v/v/m in fungus cultures.     

Inhibitory effect of 13 taxane diterpenoids from Chinese yew (Taxus chinensis var. mairei) on the proliferation of HeLa cervical cancer cells

The inhibitory effect of 13 taxanes isolated from the Chinese yew (Taxus chinensis var. mairei) on the proliferation of human cervical cancer HeLa cells were examined using an MTT assay. Four compounds having a hydrophobic cinnamate side chain showed antiproliferative activity, which may be due to increased cell permeability.     

Translocation of isopentenyl pyrophosphate for Taxol biosynthesis in suspension cultures of Taxus chinensis var. mairei

Two inhibitors of metabolite translocators, sodium pyrophosphate (NaPP) and D,L-glyceraldehyde (DLG), respectively, were added into suspension cultures of Taxus chinensis var. mairei at the early and late growth phases to investigate the translocation of isopentenyl pyrophosphate (IPP) from cytoplasm to plastids for Taxol biosynthesis. NaPP and DLG hardly affected cell biomass and viability regardless of the phases of their introduction. The Taxol content varied less when NaPP or DLG was added at day 7 but decreased obviously when NaPP or DLG was introduced at day 14. It is thus inferred that NaPP and DLG inhibited Taxol biosynthesis by blocking IPP translocation at the late growth phase, suggesting that the translocation of IPP be involved only at the late growth phase of Taxus cells.     

Microsatellite loci from Taxus chinensis var. mairei (Taxaceae), an endangered and economically important tree species in China

Taxus chinensis var. mairei is an endangered and economically important tree species in China. Four polymorphic microsatellite markers were developed and characterized using the fast isolation by amplified fragment length polymorphism (AFLP) of sequences containing repeats (FIASCO) protocol. Polymorphism of each locus was quantified for five natural populations from Hubei Province, China. The number of alleles per locus varied from 6 to 24. The ranges of observed and expected heterozygosity were 0.493–1.000 and 0.440–0.845, respectively. These are the first microsatellites reported for the family of Taxaceae and will be helpful for the ongoing population and conservation genetics research of Taxus chinensis var. mairei.     

De novo transcriptome analysis of Taxus chinensis var. mairei to identify significant pathways associated with the fruit color of this species

The goal of this study was to find significant pathways associated with fruit color in Taxus chinensis var. mairei. Two Taxus chinensis var. mairei with fruits of different colors were sequenced using high-throughput sequencing. Then, unigene assembly, unigene annotation, and classification of Clusters of Orthologous Groups of proteins (COGs) were performed. Gene ontology (GO) classification and pathway enrichment analysis for unigenes were also conducted. Protein coding sequences (CDS) were predicted. Subsequently, differentially expressed unigenes, functional enrichment pathways of differentially expressed unigenes, single nucleotide polymorphisms (SNPs), and simple sequence repeats (SSRs) were analyzed. The data of Taxus chinensis var. mairei were assembled into 45,112 unigenes with an average size of 1232 bp and an N50 of 1882 bp. The significantly enriched biological processes (BP) were metabolic processes, and cellular processes, followed by single-organism processes, and reproductive processes. Significantly enriched Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways were metabolic pathways, pathways related to the biosynthesis of secondary metabolites, and RNA transport pathways. Transition had a higher number of SNP sites than transversion. Trinucleotide had the highest SSR motif numbers. Metabolic pathways and the biosynthesis of secondary metabolites may play significant roles in determining fruit color in Taxus chinensis var. mairei. In addition, the present dataset would provide a basis for future functional genomic research on Taxus chinensis var. mairei.     

Chemistry of Chinese yew, Taxus chinensis var. mairei

Taxus chinensis var. mairei (Lemée et Lévl) Cheng et L.K. Fu is an evergreen tall tree ubiquitous to the southeastern region in China. The first chemical study on this species was published in 1987. Since then about 163 compounds including taxoids and non-taxane compounds were isolated from seeds, root, bark and leaves of this species. This review summarized the chemical investigation on T. chinensis var. mairei (Lemée et Lévl) Cheng et L.K. Fu in the past 20 years. T. chinensis var. mairei (Lemée et Lévl) Cheng et L.K. Fu was assigned as a variation of T. chinensis (Pilger) Rehd. in Flora of China. However, present chemotaxonomic data would suggest that these two plants might belong to two different chemotypes; further genetic investigation and comprehensive chemical studies on them are warranted to address this issue.     

The complete chloroplast genome sequence of Taxus chinensis var. mairei (Taxaceae): loss of an inverted repeat region and comparative analysis with related species

Taxus chinensis var. mairei (Taxaceae) is a domestic variety of yew species in local China. This plant is one of the sources for paclitaxel, which is a promising antineoplastic chemotherapy drugs during the last decade. We have sequenced the complete nucleotide sequence of the chloroplast (cp) genome of T. chinensis var. mairei. The T. chinensis var. mairei cp genome is 129,513 bp in length, with 113 single copy genes and two duplicated genes (trnI-CAU, trnQ-UUG). Among the 113 single copy genes, 9 are intron-containing. Compared to other land plant cp genomes, the T. chinensis var. mairei cp genome has lost one of the large inverted repeats (IRs) found in angiosperms, fern, liverwort, and gymnosperm such as Cycas revoluta and Ginkgo biloba L. Compared to related species, the gene order of T. chinensis var. mairei has a large inversion of ~ 110 kb including 91 genes (from rps18 to accD) with gene contents unarranged. Repeat analysis identified 48 direct and 2 inverted repeats 30 bp long or longer with a sequence identity greater than 90%. Repeated short segments were found in genes rps18, rps19 and clpP. Analysis also revealed 22 simple sequence repeat (SSR) loci and almost all are composed of A or T.     

Population characteristics of endangered species Taxus chinensis var. mairei and its conservation strategy in Shanxi, China

The conservation of the endemic, endangered, and first-class protected tree species, Taxus chinensis var. mairei (Lem. & H. Lev.) W. C. Cheng & L. K. Fu, is urgent in China. To assist the protection of this important medicinal plant species, the biological and ecological characteristics of its populations were investigated and analyzed in Shanxi. The study area, southeastern Shanxi, is a special region suitable for this species growth and development. The floristic composition, life form spectrum, and species diversity of T. chinensis var. mairei forests reflected the features of both warm-temperate and subtropical regions. Size classes in DBH and static life table analysis showed that the population structure of T. chinensis var. mairei was of the increase-type. Plant density, survivorship, and mortality decreased with increase in age. Distribution patterns of individuals were basically clumped and saplings tended to be distributed randomly. Disturbance from collection of leaves, branches, and stems for medicine was an important factor affecting population structure, dynamics, and distribution. The sex ratio of female/male and the efficiency of sexual reproduction were very low. The main way of reproduction for this species is vegetative propagation. Based on these characteristics, some suggestions, such as establishing nature reserves, improving population structure by cultivation and transplantation, developing seed gardens, and plantations, etc., for the conservation management of T. chinensis var. mairei and its forests in Shanxi are discussed.     

Comparison of Morphological and Anatomical Characteristics of <i>Taxus chinensis</i> var. <i>mairei </i>Seedlings Root under Waterlogging Stress in Different Substrates

Four different ratios of river sand, ceramic pellets, vermiculite and perlite (1:1), and field soil were selected as the substrates in this experiment, and four gradient levels of root waterlogging, half waterlogging, full waterlogging and normal were set to investigate the effects of different gradients of waterlogging stress on the root morphology of Taxus chinensis var. mairei seedlings under different substrates. In this study, the root anatomical structure of Taxus chinensis var. mairei under different waterlogging stress was observed by the paraffin section method. The roots of T. chinensis var. mairei were diarch, with no pith and resin canals. There was a large number of tannins in the pericycle of the aerial adventitious roots of seedlings adapted to waterlogging. Also, the endodermis has obvious casparian strip thickening, and there were 4-5 layers of large parenchymatous cells in the close to the inner side of the pericycle in the vascular cylinder, which could increase the storage capacity, and transport capacity of the root. Under the treatment of root waterlogging stress, the development of plant roots in the mixed substrate of vermiculite and, perlite was the earliest. Under half waterlogging stress, T. chinensis var. mairei seedlings treated with various substrates all could better adapt to the environment of waterlogging stress. Under the stress of fully waterlogging, the roots of seedlings planted in river sand substrate developed secondary growth.     

不同遮荫强度下南方红豆杉枝叶紫杉醇产量的季节变化

研究了在一个生长季节内遮荫网不同遮荫强度下对人工种植的南方红豆杉(Taxus chinensis var. mairei)枝叶生物量、紫杉醇含量和产量季节变化。结果表明,在遮荫网89％和46.4％遮光条件下,南方红豆杉枝叶生物量、紫杉醇含量及其产量随发育节律呈现明显的规律性季节变化。在遮荫网89％和46.4％遮光条件下,89％遮荫条件下南方红豆杉枝叶生物量在整个生长季节内都明显高于46.4％遮荫条件下南方红豆杉枝叶生物量;在遮荫网89％和46.4％遮光条件下南方红豆杉枝叶中紫杉醇含量在5月中旬、7月末和11月末都出现较高峰值,后者紫杉醇含量峰值都明显比前者紫杉醇的含量高;89％和46.4％遮荫网遮光条件下南方红豆杉枝叶中紫杉醇的产量都在11月末期时达到最高,后者明显高于前者。因此,生产实践中可采用46.4％遮荫网遮光,采收的最佳季节为11月末期。     

遮荫对南方红豆杉光合特性及生活史型影响

对浙江省富阳市种植的5年生南方红豆杉,89%遮荫的条件下生长的南方红豆杉与46.4%遮荫及自然光条件诱导50天的南方红豆杉的光合特性、光合色素及其生活史型的研究。结果表明:89%遮荫、46.4%遮荫和自然光的光补偿点分别为18.88,30.52和65.34 μmol·m^-2·s^-1,光饱和点分别为287.01,258.25和358.92 μmol·m^-2·s^-1,遮荫可以降低南方红豆杉的光补偿点和光饱和点,从而能够更好地利用弱光,同时提高光合速率,增强了南方红豆杉的光合能力,其中以89%遮荫的变化最明显。随着遮荫程度的增大,南方红豆杉叶片的叶绿素a、叶绿素b及类胡萝卜素等光合色素的含量均增大,叶绿素a与叶绿素b含量的比值减小,说明遮荫可以增加植物对光能的利用,尤其是增加了对蓝紫光的利用,提高了光合效能。研究还发现遮荫对南方红豆杉的生活史型有一定的影响,89%遮荫、46.4%和自然光的生活史型分别为V_0.836C_0.164,V_0.625C_0.375和V_0.772C_0.228,以89%遮荫的营养生长最为旺盛。因此,89%遮荫是这三种光照条件中南方红豆杉营养生长的最适条件。     

Taxus associated fungal endophytes: anticancerous to other biological activities

Endophytes are the plant microorganisms that flourish inside plants (endosphere) and support the growth under normal and challenging environmental circumstances. Besides biological activities, microbial endophytes are important for their contribution in adaptation and survival of the host plants under stressed environments. While various species of Taxus have been globally recognized mainly due to their anticancer property, emphasis has also been on isolation of taxol producing endophytes in view of the taxonomic (endangered) status of the species. Furthermore, Taxus associated endophytes are also being recognized for the biological activities other than taxol production. The present study reviews the diversity of Taxus associated endophytes along with their biological activities. Available literature on Taxus associated endophytes revealed that T. chinensis has received maximum attention (19.5 %), followed by T. wallichiana (18.5 %), T. baccata (16.4 %), T. cuspidata (11.3 %), and T. mairei (10.3 %); with a minimum number of reports accounted for T. globose (1.0 %), and T. celebica (2.0 %). Most fungal endophytes have been identified as ascomycetes. Basidiomycetous endophytes have been reported in a limited number of studies. No endophytes have yet been identified from three species, namely T. controta (West Himalayan yew), T. floridana (Florida yew), and T. canadensis (Canadian yew). A total of 70 diverse endophytic species have been reported from different Taxus species. Fusarium sp. (25.2 %), Aspergillus sp. (17.6 %), Alternaria sp. (11.3 %), Penicillium sp. and Cladosporium sp. (10.0 %) are the five dominant genera. Furthermore, 59.6 % of studies from the reported literature are related to taxol production by fungal endophytes, 9.6 % related to antimicrobial activity, and a combined 15.3 % related to other biological activities, i.e., antifeeding, antimitogenic, antioxidant and cytotoxic activities. Additionally, 3.8 % of fungal endophytes were not studied for any of their biological activities. Only three bacterial endophytes namely Burkholderia sp., Enterobacter sp., and Pseudomonas sp., have been recorded from Taxus along with their contribution in plant growth, antagonistic, and antioxidant activities. Isolation and characterization of microbial endophytes from Taxus plant parts is important for the associated ecology and medicinally important biological activities. It will support the much-required conservation strategy of Taxus species. Further, isolation of plant growth promoting microorganisms from Taxus will help in propagation of the species.     

Development of SCoT-Based SCAR Marker for Rapid Authentication of <Emphasis Type="Italic">Taxus Media</Emphasis>

Taxus media is an important species in the family Taxaceae with high medicinal and commercial value. Overexploitation and illegal trade have led T. media to a severe threat of extinction. In addition, T. media and other Taxus species have similar morphological traits and are easily misidentified, particularly during the seedling stage. The purpose of this study is to develop a species-specific marker for T. media. Through a screening of 36 start codon targeted (SCoT) polymorphism primers, among 15 individuals of 4 Taxus species (T. media, T. chinensis, T. cuspidate and T. fuana), a clear species-specific DNA fragment (amplified by primer SCoT3) for T. media was identified. After isolation and sequencing, a DNA sequence with 530 bp was obtained. Based on this DNA fragment, a primer pair for the sequence-characterized amplified region marker was designed and named MHSF/MHSR. PCR analysis with primer pair MHSF/MHSR revealed a clear amplified band for all individuals of T. media but not for T. chinensis, T. cuspidate and T. fuana. Therefore, this marker can be used as a quick, efficient and reliable tool to identify T. media among other related Taxus species. The results of this study will lay an important foundation for the protection and management of T. media as a natural resource.     

Infraspecific variability ofJuniperus L. (Cupressaceae) based on monoterpenoid compositions

The compositions of leaf monoterpenoids from 11 species in the Juniperus section (Juniperus chinensis var. chinensis,J. virginiana, J. communis var. montana,J. rigida, J. chinensis var. globosa,J. chinensis var. sargentii,J. chinensis ‘Kaizuka Variegata’,J. squamata ‘Wilsonii’,J. x media ‘Shimpake’,J. x media ‘Plamosa Aurea’, andJ. squamata ‘Aloderi’) were comparatively analyzed by GC-MS. Of the 24 compounds identified, α-pinene, myrcene, limonene, terpinolene, and bornyl acetate were common to all, but particular combinations differed remarkably among taxa. The simplest composition (eight compounds) was found in.J, chinensis var. chinensis; the most complex (19 compounds), inJ. x media ‘Shimpake’. Cluster analysis generated four distinctive clades within the Juniperus section. The minimum spanning network revealed thatJ. squamata ‘Wilsonii’ andJ. x media ‘Shimpake’ were most similar in their chemical makeup.     

南方红豆杉生长发育及其紫杉醇含量与环境因子的关系

研究了人工种植的3年生南方红豆杉的生长发育特征及其枝叶中紫杉醇的含量,并观测了其生长环境因子的动态特征。通过对南方红豆杉的生长发育特征以及其枝叶中紫杉醇含量与生长环境因子进行相关分析发现,南方红豆杉生长发育与空气和土壤湿度呈极显著的正相关,与空气和土壤温度、土壤N和K含量、土壤有机质和土壤紧实度呈正相关,与净辐射和土壤P含量呈负相关,但相关关系不显著,说明高温、高湿以及遮荫有利于南方红豆杉的生长,且对土壤P含量有较高需求;南方红豆杉枝叶中紫杉醇含量与空气和土壤湿度、土壤N、P和K含量呈负相关,与净辐射、空气和土壤温度、土壤有机质和土壤紧实度呈正相关,但相关关系均不显著,说明高温、高光强和干旱促进南方红豆杉枝叶中紫杉醇含量的增加,且紫杉醇含量的增加对土壤中N、P和K含量有一定的需求,尤其是N和K,对P需求不大;南方红豆杉生长发育与其枝叶中紫杉醇含量呈负相关,相关关系也不显著,推测紫杉醇可能是南方红豆杉的防御物质,以促使南方红豆杉抵御高温、干旱和高光强等不良环境。     

Screening of taxol-producing endophytic fungi from <Emphasis Type="Italic">Taxus chinensis</Emphasis> var. <Emphasis Type="Italic">mairei</Emphasis>

A total of 38 endophytic fungus strains were isolated from Taxus chinensis var. mairei by the aseptic technique. Genomic DNA was extracted from isolated endophytic fungi and subjected to polymerase chain reaction (PCR) analysis for the presence of the Taxus taxadiene synthase (TS) gene, a rate-limiting enzyme gene in the taxol biosynthetic pathway. Twelve out of 38 isolated endophytic fungus strains showed PCR positive for the ts gene. Subsequently, taxol and its related compounds were extracted from culture filtrates and mycelia of the PCR positive strains, separated by column chromatography, and analyzed by High Performance Liquid Chromatography and Mass Spectrum. The analysis result showed that 3 strains could produce taxol and its related compounds at the detectible level. This study indicates that molecular detection of the ts gene is an efficient method for primary screening of taxol or its related compound-producing endophytic fungi, which can improve prominently screening efficiency. Published in Russian in Prikladnaya Biokhimiya i Mikrobiologiya, 2007, Vol. 43, No. 4, pp. 490–494. The text was submitted by the authors in English.     

Apoptotic cell death in suspension cultures of Taxus chinensis var. mairei

Apoptotic cell death was observed in suspension cultures of Taxus chinensis var. mairei under normal cultivation conditions by using microscopy, total DNA agarose gel electrophoresis and in situ end-labeling of fragmented DNA. The morphological and biochemical changes of cells occurred mainly in the non-dividing cell clusters, indicating that the T. chinensis cells died mainly by apoptosis. There exists a close relationship between cell apoptosis and Taxol formation. Taxol concentration increased with the increase in content of apoptotic cells and reached a maximum (14.2 mg l^–1) after 23 days of culture, corresponding to a maximum ratio of apoptotic to total cells of about 13%.