Combining ectomycorrhizal fungi and plant growth-promoting rhizobacteria to enhance salt tolerance of Metasequoia glyptostroboides

Plant growth and productivity are negatively affected by soil salinity.This study investigated the effects of the rhizosphere-promoting bacterium,Bacillus paramycoides JYZ-SD5,and the ectomycorrhizal fungus,Schizophyllum commune Be,on the growth of Metasequoia glyptostroboides under salt stress.Changes in biomass,root growth,root ion distribution and in vivo enzyme activities were determined under different treatments(Be,JYZ-SD5,and Be+JYZ-SD5).The results show that all inoculations increased chlorophyll content,shoot length and root diameter with or without salt stress,and the effect of Be+JYZ-SD5was the strongest.JYZ-SD5 and Be+JYZ-SD5 treatments significantly increased root length,surface area,bifurcation number,tip number,main root length and diameter under salt stress.Normal chloroplast structures developed under both single and double inoculations.Relative to the control,root activities of M.glyptostroboides in the Be,JYZSD5,and Be+JYZ-SD5 treatments increased by 31.3%,17.2%,and 33.7%.All treatments increased the activities of superoxide dismutase(SOD),peroxidase(POD),Na^(+)-K^(+)-ATPase and Ca^(2+)-Mg^(2+)-ATPase.The strongest effect was by Be+JYZ-SD5.Analysis of root ion distribution showed that,under salt stress,Na^(+)and K^(+)decreased and were concentrated in the epidermis or cortex.Na/K ratios also decreased.The Be+JYZ-SD5 treatment increased betaine by 130.3%and 97.9%under 50 mM and 100 mM salt stress,respectively.Together,these changes result in the activation of physiological and biochemical processes involved in the mitigation of salinity-induced stress in M.glyptostroboides.

Anti-listerial synergism of leaf essential oil of Metasequoia glyptostroboides with nisin in whole,low and skim milks

Objective:To examine the individual and synergistic anti-listcrial effect of nisin and leaf essential oil of Metasequoia glyptostroboides(M.glyptostroboides) against one of the leading foodhorne pathogens Listeria monocytogenes(L.monocytogenes) ATCC 19116 in milk samples.Methods:The whole(8%),low(1%) and skim(no fat content) milk samples were inoculated with L.monocytogenes ATCC 19116 along with leaf essential oil of M.glyptostroboides or nisin alone as well in eomhinations.Results:In this study,the leaf essential oil at the concentrations of 2% and 5% revealed strong anti-listerial effect against L.monocytogenes ATCC 19116 in all categories of milk samples.Nisin at the concentrations of 250 and 500 IU/mL displayed a strong inhibitory effect against ATCC 19116 as compared to the control group.Additionally,synergistic combinations of leaf essential oil(1%) and nisin(62.5,125.250 and 500 IU/mL) also had a remarkable anti-listerial synergism in all the tested milk samples including whole,low and skim milk after 14 days.Conclusions:As a major finding,the leaf essential oil of M.glyptostroboides might he a useful candidate for using in food industry to control the growth of foodhorne pathogenic bacteria as confirmed by its potent anti-listerial Synergistic effect with nisin against L.monocytogenes ATCC 19116 in different milk samples.

Genetic variation in progenies of 23 relic trees of Metasequoia glyptostroboides in their original habitat

Seeds of 23 relic Metasequoia glyptostroboides trees were randomly collected from their original, natural opulations found in Lichuan City, Hubei Province, China. The seed characteristics and growth of their three-year-old rogenies were investigated. Results show that the average intra-family coefficient of variation （CV） in height growth of ：he 23 three-year-old families was 14.53%, slightly higher than that of the inter-family CV （14.02%）. Correspondingly, hese two parameters for root-collar diameter growth were 15.44% and 13.15%. The 23 relic mother trees were ignificantly differed in their general combining abilities （GCA） and relative effect values of GCA （RGCA） of height growth and root-collar diameter growth over the three years. Both height and root-collar diameter growth had high amily heritability in this seedling stage. After eliminating two inferior families, i.e., 15 and 46, 2.21% of genetic gain in he ght growth was obtained and 1.50% in root-collar diameter growth. It was also significantly different in crown size and number of branches among the 23 three-year-old seedling families. From a cluster analysis we concluded that eight families, i.e., 23, 28, 29, 30, 31, 33, 35 and 44 displayed superior GCA and RGCA in both height and root-collar diameter growth. Therefore, these relic mother trees displayed their potential as parents in seed orchards. With the exception of family 23, the other seven superior families were all collected at Guihua Village, Zhonglu Town, Lichuan City, which has been identified as an optimum district of M. glyptostroboides for seed collection.

Three Novel Endophytic Fungi as Potential Candidates Producing Bioactive Metabolites Isolated from Metasequoia glyptostroboides

[Objectives]This study was conducted to isolate and investigate the endophytic fungi with antimicrobial activity from the healthy leaves and bark of Metasequoia glyptostroboides Hu&W.C.Cheng regarded as an important medicinal plant in China.[Methods]Endophytic fungi with visible antimicrobial activity were isolated from the healthy leaves and bark of M.glyptostroboides,and primarily screened using Escherichia coli and Staphylococcus aureus as test microorganisms.[Results]Three isolates with relatively higher antimicrobial activity,labeled as MG-01,MG-02,MG-09,were selected for study.Molecular and morphological analysis indicated that the three isolated endophytes belong to Penicillium sp.The ethyl acetate(EtOAc)extracts of the three endophytes were obtained and subjected to the disk diffusion assay,using E.coil,S.aureus,Pseudomonas aeruginosa,Enterococcus faecalis,Monilia albicanas,and Bacillus subtilis as test microorganisms.The minimum inhibitory activity(MIC)of the endophyte with relatively highest antimicrobial activity was determined using two-fold broth micro-dilution method.All the endophytes showed a broad spectrum antimicrobial activity.Among the three endophytes,the strain MG-09 exhibited relatively highest antimicrobial activity with the zones of inhibition(ZOI)ranging from 14.15 to 24.07 mm and MIC ranging from 416.67 to 65.11μg/ml.[Conclusions]The endophytic fungi colonized in M.glyptostroboides are capable of producing effective antimicrobial compounds which may be a potential source for drug discovery and biocide production.

N-Exponential Fertilization Could Affect the Growth and Nitrogen Accumulation of Metasequoia glyptostroboides Seedling in a Greenhouse Environment

Metasequoia glyptostroboides(M.glyptostroboides)is a unique plant species related to relic flora in China.It plays a positive role in afforestation and its long-term protection with high paleoclimate research value.However,due to the nutrients-supply deficiency,it is a big challenge to cultivate the high-quality seedlings of M.glyptostroboides.In this study,a pot experiment in a greenhouse environment was carried out to identify the effect of N-exponential fertilization on the growth and nutrient distribution of M.glyptostroboides seedling.The M.glyptostroboides rooted seedlings with 12-month growth were chosen.Different N fertilizer levels with conventional fertilization(CF:5.0 g seedling^(−1)),exponential fertilization including EF1,EF2,EF3 and EF4 were determined.The relevant growth indexes were measured after 210-day growth.The results indicated that non-significant differences in seedlings’height and ground diameter were found among the above treatments(P>0.05);At the same time,N-exponential fertilization promoted the M.glyptostroboides’s biomass in different organs(P<0.05),with the maximum total biomass under EF3 treatment.The N accumulation in root and stem of the N-exponential fertilization treatments were increased in to some extent(P<0.05).The maximum N accumulation was also found under EF3 treatment.Therefore,steady-state nutrition and superior growth performance of M.glyptostroboides could be obtained by N-exponential fertilization of 5.0 g cutting^(−1).

Protection of Metasequoia glyptostroboides Hu et Cheng in China

Metasequoia （Metasequoia glyptostroboides Hu et Cheng） is an important but endangered tree species. In a sense, the course of protection of the metasequoia can be considered a miniature of the history of the protection of rare Chinese plants over the past 50 years. The approval and establishment of the National Class Nature Reserve for metasequoia is a milestone in this course. A thorough understanding of this course is beneficial to design the correct future protection plans and for taking effective measures. In this paper the entire protection course of metasequoia is reviewed, three detailed measures are proposed according to the present situation and existing problems and discussed as follows. 1） We should reinforce unceasingly the establishment of National Nature Reserves of metasequoia, put emphasis on the protection of the original natural mother trees （ONMTs） of metasequoia, design far- sighted projects to protect the ONMTs and strictly implement and enforce the regulations or laws regarding the protection of meta- sequoia. The establishment and functions of the organization should meet the demands of protecting the ONMTs and developing metasequoias. The budget should support and the quality of the personnel assigned to this task should satisfy the protection and de- velopment of the ONMTs. We should also promote the complete protection of the ONMTs at the same time and strengthen the pro- tection and restoration of the ONMTs and their ecosystem in the Xiaohe valley, Lichuan County, Hubei Province, one of the natural concentration areas of ONMTs in the world. 2） A gene bank should be established for comprehensive protection of the genes of ex- isting original mother trees of metasequoia. 3） The key is to study the theoretical fundamentals of restoration and re-establishment of natural vegetation of metasequoia in the Xiaohe valley to harmonize the relation among environmental departments, forestry sectors, academies of sciences and other educational or research units, to carry out scientific research and strengthen academic exchanges to promote jointly the protection and utilization of metasequoia and other natural resources in China.

Atomic force microscopic observation on substructure of pollen exine in Cedrus deodara and Metasequoia glyptostroboides

The substructure of pollen exine in Cedrus deodara (Roxb.) Loud, and Metasequoia glyptostroboides Hu et Cheng has been examined with an atomic force microscope (AFM). The results indicate that the exine substructure units containing sporopollenin in two species are similar in shape, which are granular, but slightly different in size. In Cedrus the substructure unit of pollen exine appears to be 56-99 nm long and 42-74 nm wide, while in Metasequoia it appears to be 81-118 nm long and 43-98 nm wide. It has been observed that the subunits of pollen exine in Cedrus arranged tightly to form short-rod-like or spheroidal pollen exine units, several or more than ten of which formed an island-like structure. There are various spaces among these island-like structures which are interconnected to occupy the entire pollen exine. In Metasequoia, the subunits of pollen exine also arranged tightly with a distribution tendency of cluster of 3-10, however, no obvious boundary exists among these clusters. From our

孑遗植物水杉种子萌发的生理生态特性研究

从种子活力和抗氧化酶活性两个方面研究了温度和光照对孑遗植物水杉 (Metasequoiaglyptostroboides)种子萌发的生理生态特性的影响。实验结果表明 ,萌发温度对水杉种子的活力和抗氧化酶活性有较大影响 ,在 19-2 8℃范围内 ,种子的发芽率、发芽速率、活力指数、脱氢酶及超氧化物歧化酶 (SOD)、过氧化物酶 (POD)和过氧化氢酶 (CAT)等抗氧化酶活性均较高 ,其中在 2 4℃条件下最高 ;当温度低于 15℃或者高于 32℃时 ,种子活力很低 ;10℃条件下种子几乎不萌发。可以认为 ,水杉种子萌发的适宜温度范围为 19- 2 8℃ ,最适温度为 2 4℃。光照萌发试验表明 ,6 5 μmol·m-2 ·s-1(12h/d)光照对水杉种子的萌发有较大的抑制作用 ,黑暗条件更适于水杉种子的萌发。自然状态下水杉种子质量很差 ,空瘪率超过 90 % ,加之产地春季温度不适于水杉种子的萌发 ,这两者可能是限制水杉种群更新的重要因素。这也许是水杉种群中缺乏幼苗和幼树的原因之一。

水杉的保护历程和存在的问题

水杉(Metasequoia glyptostroboides)的保护过程在某种程度上可以说是新中国成立50年来中国珍稀植物保护过程的一个缩影。2003年,以保护水杉为主要目的而建设的湖北省国家级星斗山自然保护区是我国水杉保护和发展中的里程碑,对于这一过程的了解有助于今后正确地制订就地和迁地保护计划和采取有效的行动。本文对水杉保护历程进行了全面回顾,针对水杉原产地的保护现状和存在的问题,提出四条具体的保护措施:(1)加强以水杉保护为主要目的的国家级自然保护区建设,建设重点应放在对水杉原生母树及其生境的保护。保护水杉原生母树及其生境要有长远的规划,机构的设置和功能以及人员的素质和经费要能支持水杉原生母树及其生境保护和发展的需要;(2)要把有关水杉保护的法规和措施落到实处;(3)建立基因保存林,对现有水杉原生母树基因进行全面保存;(4)积极协调环保、林业、科学院以及其他教育科研单位的关系,加强在这一领域的分工协作,开展科学研究,加强学术交流,共同促进水杉的保护和利用。

水杉种子中总黄酮含量及其清除自由基能力分析

用紫外分光光度法,以芦丁作对照,测定水杉(Metasequoia glyptostroboides)种子中的总黄酮含量,以BHT和维生素C作对照,比较其对DPPH自由基的清除能力。结果表明,水杉种子中总黄酮含量为34.7 mg/g,其清除DPPH自由基的能力随质量浓度升高而增强,达到EC=145.6μg/mL,低浓度时低于BHT,随着浓度增高,高于相同质量浓度的BHT,低于维生素C,但在400μg/mL时,与维生素C持平。水杉种子中黄酮有较高利用价值,作为天然抗氧化剂有良好的应用价值。

水杉药学研究概况

在广泛文献检索基础上,对水杉的成分、药理及临床应用价值等进行了综述,为水杉的深入开发利用提供参考资料。

具有抗菌活性的水杉内生真菌的分离及筛选

从水杉(Metasequoia glyptostroboides Hu et Cheng)树皮、叶片中分离筛选得到了17株形态各不相同的内生真菌,采用对峙培养法筛选对植物病原真菌,层出镰孢菌(Fusarium proliferatum)﹑禾谷镰刀菌(Fusarium graminearum)﹑玉米大斑病菌(Setosphaeria turcica)﹑玉米小斑病菌(Helminthosporium maydis)具有拮抗作用的菌株,筛选得到了1株对以上4种病原菌具有广谱抗菌活性的菌株,命名为SS-17。通过对SS-17菌株的形态特征观察及ITS分子鉴定,初步鉴定为黑附球菌(Epicoccum nigrum)。

Influencing factors and growth state classification of a natural Metasequoia population

By analyzing the importance of influencing factors and conducting a comparative study of the effects of different sorting algorithms, a new method is proposed that is suitable for classifying the growth state of a natural Metasequoia glyptostroboides Hu and W.C. Cheng population. We studied 2817 M. glyptostroboides trees over 100 years old and analyzed their growth state by measuring 15 factors from stumpage, site condition, and environmental data. The dimensionality of all factors were reduced using the random forest algorithm, and we classified the remaining factors using the following algorithms: random forest, back-propagation(BP) neural networks, and support vector machine(SVM). The applicability of each sorting algorithm was analyzed. When all the d factors are used for classification and modeling, the model's overall accuracy,kappa coefficient and test accuracy were 85.5%, 0.739 and 85.8%, respectively. By reducing the dimensionality of the factors using the random forest algorithm, 11 factors most strongly influenced the classifications of the growth state of the Metasequoia population: diameter at breast height,height, crown width, age from stumpage data; longitude,latitude, elevation, slope aspect, gradient and slope position from the site condition data; and the edge of the field from the environmental data. For classifying the Metasequoia population, the random forest algorithm has the highest overall accuracy at 87.2%, which is 3.4 and 2.3% higher than the BP neural networks and SVM algorithms,respectively. The SVM algorithm is superior to the random forest algorithm with respect to classifying the state of mortality. The combination of the random forest and SVM algorithms and their combined information can be used to classify and predict the growth state of this natural M.glyptostroboides population to provide a scientific basis for its effective protection.

The Metasequoia genome and evolutionary relationships among redwoods

Redwood trees(Sequoioideae),including Metasequoia glyptostroboides(dawn redwood),Sequoiadendron giganteum(giant sequoia),and Sequoia sempervirens(coast redwood),are threatened and widely recognized iconic tree species.Genomic resources for redwood trees could provide clues to their evolutionary relationships.Here,we report the 8-Gb reference genome of M.glyptostroboides and a comparative analysis with two related species.More than 62%of the M.glyptostroboides genome is composed of repetitive sequences.Clade-specific bursts of long terminal repeat retrotransposons may have contributed to genomic differentiation in the three species.The chromosomal synteny between M.glyptostroboides and S.giganteum is extremely high,whereas there has been significant chromosome reorganization in S.sempervirens.Phylogenetic analysis of marker genes indicates that S.sempervirens is an autopolyploid,and more than 48%of the gene trees are incongruent with the species tree.Results of multiple analyses suggest that incomplete lineage sorting(ILS)rather than hybridization explains the inconsistent phylogeny,indicating that genetic variation among redwoods may be due to random retention of polymorphisms in ancestral populations.Functional analysis of ortholog groups indicates that gene families of ion channels,tannin biosynthesis enzymes,and transcription factors for meristem maintenance have expanded in S.giganteum and S.sempervirens,which is consistent with their extreme height.As a wetland-tolerant species,M.glyptostroboides shows a transcriptional response to flooding stress that is conserved with that of analyzed angiosperm species.Our study offers insights into redwood evolution and adaptation and provides genomic resources to aid in their conservation and management.

水杉40S核糖体蛋白S8基因的克隆及生物信息学分析

水杉(Metasequoia glyptostroboides)是全世界著名的孑遗植物,而核糖体蛋白又是细胞中参与蛋白质合成及调控基因表达的一种重要蛋白。因此,为了进一步展开对水杉核糖体蛋白基因的结构和功能研究,本研究在构建水杉全长转录组文库的基础上,克隆了一个40S核糖体蛋白S8基因(MgRPS8),借助于生物信息学手段对其进行了分析。结果显示:该基因c DNA全长为1 497 bp,编码221个氨基酸;编码蛋白分子量为25.1 kD,等电点为10.39,总平均吸水值为-0.968;含有19个常见功能位点;二级结构包括5个螺旋区、7个折叠区和一些无规则卷曲区;三级结构同源比对的结果进一步证实该基因的编码蛋白为真核生物核糖体蛋白S8;系统发育树构建的结果表明,该基因与日本柳杉的40S核糖体蛋白S8基因具有较近的亲缘关系。本研究结果可为水杉及其它物种的核糖体蛋白基因的结构和功能研究提供分子基础参考。

基于RNA-seq技术的水杉半胱氨酸合成酶基因的克隆分析

水杉(Metasequoia glyptostroboides)是世界著名的孑遗植物,但对其在分子水平研究的报道极少。为了获得与耐逆境胁迫有关的半胱氨酸合成酶基因,本研究以水杉原生种为实验材料,基于高通量转录组测序(high-throughput mRNA sequencing,RNA-seq)技术从全长转录组文库中克隆了一个半胱氨酸合成酶基因(MeGl-CSase),并对该基因作了进一步的生物信息学分析。结果表明,MeGl-CSase基因的全长cDNA为1702 bp,编码长度为465个氨基酸的半胱氨酸合成酶蛋白;该蛋白的分子量为49471.35 Da,总平均吸水值为-0.071,等电点为7.95;含有39个常见功能位点;二级结构包括11个折叠区、21个螺旋区以及一些卷曲区;三级结构预测结果进一步证明MeGl-CSase基因编码的产物是真核生物半胱氨酸合成酶蛋白。本研究结果可为水杉及其它物种的半胱氨酸合成酶蛋白基因的结构和功能研究提供参考。

水杉长链非编码RNA分析

水杉(Metasequoia glyptostroboides)是中国著名的一级保护植物,素有"活化石"之称。为了更好地保护和利用这一重要种质资源,本研究以水杉为实验材料,在全长转录组测序的基础上,利用四个软件对61057个Unigenes进行了蛋白编码潜能预测,最终获得3385个长链非编码RNA(long non-coding RNAs,lncRNAs)分子。进一步对这些lncRNAs的长度和数量作统计分析,结果显示,最短为200 nt,最长为11134 nt,平均长度为1956 nt;大部分lncRNAs的长度介于200~2000 nt,占总量的59.5%;在2001~4000 nt的长度范围内,也发现较多的lncRNAs,达到总量的32.2%;在长度为4001~6000 nt的范围内,只有7.6%;长度超过6000 nt的lncRNAs很少,总共只有25个(占总量的0.7%)。本研究结果为水杉资源的保护和利用研究提供了一些分子依据,为其它植物在相关领域的研究提供了一些参考。

水杉泛素结合酶基因的克隆

水杉(Metasequoia glyptostroboides)是著名的杉科孑遗植物,对水杉的分子遗传研究在其资源保护和利用方面具有重要意义。本研究以水杉为实验材料,在全长转录组测序的基础上克隆了一个泛素结合酶基因(MgUC-E2),并进一步对该基因作了生物信息学分析。结果表明:MgUC-E2基因cDNA编码链全长为691 nt,可以编码长度为148个氨基酸的泛素结合酶。该酶蛋白的分子量为16545.09 kD,等电点为7.72,平均吸水值为-0.343;含有15个磷酸化修饰位点和5个常见功能位点;在预测的二级结构中共包括5个螺旋区、4个折叠区和一些无规则卷曲区;三级结构分析的结果进一步证实MgUC-E2基因就是编码泛素结合酶。本研究结果可为水杉及其它相关物种的泛素结合酶基因的结构及功能研究提供一些参考。

基于全长转录组测序的水杉SSR序列分析

水杉是中国著名的一级保护植物,为了充分开发和利用这一重要种质资源,本研究在水杉全长转录组测序的基础上,对水杉SSR(Simple sequence repeat)序列进行了详细序列分析。共发现SSR序列16227个,其中以单核苷酸为重复单位的最多,达到10290个,长度介于10~50 nt的为绝大部分,达到总数的96.1%,其余为51~315 nt,仅占3.9%。以二、三、四、五和六核苷酸为重复单位的SSR序列分别有1156、1789、25、11、54个,长度范围分别于介于12~58 nt、15~60 nt、20~44 nt、25~30 nt和30~36 nt。此外,还发现有2902个复杂SSR序列,长度介于21~4760 nt。该结果为水杉SSR分子标记开发和水杉基因资源的保护利用提供了分子依据。

水杉核糖体蛋白基因MeGl-RPL10的克隆及生物信息学分析

水杉(Metasequoia glyptostroboides)是中国有名的杉科孑遗植物,但是在分子水平的研究极少。本研究以水杉原生种为植物材料,基于三代测序技术的全长转录组文库克隆了一个核糖本蛋白基因(MeGl-RPL10)。对该基因作进一步的生物信息学分析,结果表明:MeGl-RPL10基因的全长cDNA为972 bp,编码长度为222个氨基酸的核糖体蛋白L10;该蛋白的分子量为25081.21 kD,等电点为10.30,总平均吸水值为-0.497;含有8个常见功能位点;二级结构包括4个螺旋区、9个折叠区和一些无规则卷曲区;三级结构分析的结果进一步证实MeGl-RPL10基因的编码蛋白为真核生物核糖体蛋白L10。本研究结果可为水杉及其它物种的核糖体蛋白基因的结构和功能研究提供参考。