Effect of the biocontrol bacterium <Emphasis Type="Italic">Bacillus amyloliquefaciens</Emphasis> on the rhizosphere in ginseng plantings

Panax ginseng is an important medicinal herb due to its ability to strengthen the human immune system. However, due to the increasing needs of ginseng in medicine, the continuous cropping of ginseng has become more common and has resulted in increased problems with fungal decay. Thus, chemical fungicides are commonly used in ginseng plantings, which have caused fungicide residue problems. As an alternative control measure, biocontrol bacteria can be used to manage fungal pathogens. Additionally, these bacteria are environmentally friendly and can also improve stress tolerance in plants. In this study, an antifungal bacterial strain, TB6, that possesses ACC deaminase activity was isolated from the rhizosphere of ginseng plants. This strain was identified as Bacillus amyloliquefaciens. TB6 was applied to 2-year-old ginseng seedlings for a 2-year period, and its impact on the soil rhizosphere was evaluated. The results revealed that strain TB6 decreased fungal abundance and diversity; improved urease, catalase, and phosphatase activities; and decreased the cellulase activity of the rhizosphere soil. In addition, strain TB6 also promoted root growth and increased the fresh weight of ginseng roots, in addition to increasing polyphenol oxidase and catalase activities. These results may have practical implications for the use of biocontrol bacteria in ginseng plantings.     

不同树叶凋落物对人参土壤理化性质及微生物群落结构的影响

树种选择是林下山参护育成败的关键,研究树叶凋落物对人参土壤养分、微生物群落结构组成的影响,旨在为林下山参护育选择适宜林地及农田栽参土壤改良提供科学依据和理论指导。通过盆栽试验,研究添加5.0 g色木槭Acer mono.Maxim.var.mono(A)、赤松Pinus densiflora Sieb.et Zucc.(B)、胡桃楸Juglans mandshurica Maxim.(C)、紫椴Tilia amurensis Rupr.(D)、蒙古栎Quercus mongolica Fisch.ex Ledeb.(E)树叶凋落物到土壤中,种植人参(Panax ginseng C.A.meyer)后研究土壤理化性质以及微生物群落结构的变化。结果表明:添加不同树叶处理后人参土壤性质发生改变,土壤p H值显著高于对照土壤5.91(P0.05),土壤全氮、速效氮磷、微生物碳氮在所有树叶处理中显著增加(P0.05),而土壤容重、速效钾和C/N在添加树叶处理中降低。18个土壤样品基因组,经16S和ITS1测序分别得到6064和1900个OUTs。其中细菌涵盖了42门、117纲、170目、213科、225属,真菌涵盖了24门、98纲、196目、330科、435属。不同树叶处理人参土壤细菌和真菌地位发生改变,细菌Proteobacteria是树叶分解的关键微生物,添加树叶后其多样性显著高于对照(P0.05)。而细菌Bacteroidetes和真菌Basidiomycota可能是区别阔叶林和针叶林树种的关键微生物,针叶林中含量显著低于阔叶林(P0.05),而真菌Ascomycota是针叶林分解的关键微生物。进一步从不同分类水平上得到特定树叶凋落物的特异细菌和真菌。典型相关分析(CDA)表明细菌Bacteroidetes、Chloroflexi、Actinobacteria及真菌Basidiomycota、Zygomycota、Chytridiomycota及Ascomycota的位置及多样性的改变均与土壤因子SMBN、TN、AP、SOC、AK、C/N、p H有关。综上所述,添加不同树叶后不仅提高土壤微生物量碳氮、改善土壤理化性质,同时改变微生物群落结构组成,不同树叶处理土壤理化性质不同导致人参土壤微生物组成的差异,本结果对于林下参选地和农田栽参土壤微生物改良具有理论指导作用。     

Defense response mechanisms of ginseng callus cultures induced by Yersinia pseudotuberculosis, a human pathogen

The effect of Yersinia pseudotuberculosis, the bacterial pathogen affecting humans and animals, on growth of ginseng (Panax ginseng C.A. Mey) cell cultures was studied. The bacteria strongly induced the expression of phenylalanine ammonia-lyase and β-1,3-glucanase, the proteins encoded by the defense-related genes of ginseng and inhibited the normal ginseng callus growth but did not affect the resistant cell cultures. The thermostable and thermolabile protein toxins of these bacteria are lethal to mice when induced parentherally, and they also induced the expression of the defense-related genes in ginseng callus cultures. At the same time, the ginseng cells completely suppressed the bacterial cell growth. These data suggest that the ginseng cells recognized the yersinia and developed the immune response to this pathogen. The interaction between the ginseng cells and Y. pseudotuberculosis is similar to the hypersensitive response of plants to plant pathogens.     

西洋参根残体对自身生长的双重作用

无论在自然生态环境还是在人工农田环境下,植株残体进入土壤后都会对土壤的物理化学性质以及后茬植物的生长产生重要影响。西洋参（Panax quinquefolium L.）为人参属多年生名贵药材,在栽培生产中存在严重的连作障碍问题。为了探明秋后残留在土壤中的须根降解产物对来年植株生长的影响,以及收获后残留在田间的根茬对连作西洋参生长的作用,本实验以3年生西洋参苗为研究对象,采用室内水培试验以及田间盆栽试验,通过添加西洋参根的粉碎物模拟根残体,测定其对西洋参生长的影响。水培试验中全营养液中分别添加0.02 mg/mL、0.1 mg/mL、0.5 mg/mL西洋参根粉碎物,处理后每隔5天测定植株叶片展开情况、株高、冠幅等生长指标。盆栽试验在土壤中添加0.1 mg/g根粉碎物,于栽种后1-2个月测定西洋参叶片展开情况、株高、冠幅等生长指标;水培及盆栽试验均于展叶期、现蕾期、结果期测定地上部及地下部生物量。采用高效液相色谱法（HPLC）测定根围土壤中8种酚酸类化合物的含量。试验结果表明,水培溶液中添加0.02-0.5 mg/mL根残体,可显著抑制西洋参自身地上部分生长,推迟展叶期,结果期生物量降低14.9%-45.0%;对地下部分的影响主要表现为在展叶期显著促进须根生长(p<0.05)。与水培试验相比,盆栽土壤中添加0.1 mg/g根残体同样导致西洋参展叶期推迟;不同的是处理组的地上、地下部及须根的平均生物量均高于对照。另外,添加根残体后盆栽西洋参根围土壤中丁香酸、香草醛、p-香豆酸、阿魏酸等酚酸类化感物质含量下降49.1%-81.4%,但作为逆境信号物质的水杨酸含量升高59.9%。以上结果可以初步确认根残体对西洋参早期生长具有自毒和促进的双重作用,表现为抑制地上部分生长,导致生物量显著下降;同时在生长早期促进须根生长;但在田间环境下,自毒作用可能受根残体降解速度以及土壤对降解产物吸附的影响有所减弱,使促进作用更为明显。     

基于高通量测序技术的不同年代公园绿地土壤细菌多样性

【背景】细菌多样性对绿地土壤生态功能有重要作用,但不同年代公园绿地土壤的细菌多样性尚未见相关报道。【目的】研究北京市不同年代公园绿地土壤细菌多样性和群落结构特征。【方法】利用IlluminaMiSeq测序技术,分别对北京市代表性古典公园和现代公园绿地土壤细菌群落多样性进行分析。【结果】北京市公园绿地土壤细菌群落共划分为45个已知的菌门,其中变形菌门、酸杆菌门、绿弯菌门和放线菌门为优势细菌群。土壤细菌群落α多样性分析结果表明,古典公园和现代公园的土壤细菌多样性存在差异,表现为古典公园的丰富度和多样性都高于现代公园。此外,土壤细菌群落相似性分析和主坐标分析都表明古典公园和现代公园的土壤细菌群落结构存在显著差异。冗余分析表明,对土壤微生物群落结构产生显著影响的环境因子分别为土壤含水量、有机质和全氮,其它土壤环境因子无统计学意义。首次引入公园年代作为影响因子进行冗余分析的研究结果表明,公园年代为影响公园细菌群落多样性的重要因子。【结论】不同年代公园绿地土壤细菌群落结构和物种多样性具有显著差异,随着公园年代的增加,土壤肥力和微生物多样性增加,绿地生态系统更稳定,可通过制定不同的绿地管理措施改变公园绿地土壤环境,进而优化土壤细菌群落结构,促进土壤碳氮养分循环,提高土壤肥力。     

人参种植对林地土壤细菌群落结构和代谢功能的影响

揭示人参种植对土壤微生物群落结构和代谢功能的影响,对防治人参连作障碍具有重要的理论意义。利用高通量测序技术研究了林地和由林地开垦耕种人参3a和4a后土壤微生物群落结构和代谢功能的变化。结果表明,变形菌门(Proteobacteria)、放线菌门(Actinobacteria)、酸杆菌门(Acidobacteria)、疣微菌门(Verrucomicrobia)、绿弯菌门(Chloroflexi)是林地和人参种植土壤微生物的主要优势类群。林地开垦种植人参后,土壤放线菌门和疣微菌门的相对丰度显著增加,土壤酸杆菌门的相对丰度显著降低;而土壤微生物多样性指数明显降低。除趋势对应分析(DCA)显示不同种植a限人参的土壤微生物群落结构存在明显差异。Tax4Fun功能预测表明,人参种植后土壤微生物的萜类化合物和聚酮类化合物代谢与信号转导功能的相对丰度显著降低,而膜转运功能的相对丰度增加。典范对应分析(CCA)和Partial Mantel Test分析表明,土壤速效钾、全钾含量和土壤pH值是影响土壤微生物群落结构的重要因子。因此,林地开垦种植人参对土壤微生物群落结构、多样性以及代谢功能产生了显著影响,土壤速效钾、全钾含量和pH值是影响土壤微生物群落结构的重要因素。     

不同年限对栽参土壤生物活性的影响

通过对不同年限栽参土壤微生物功能多样性和土壤酶活性的动态变化分析,探究人参生长年限与微生物碳源利用、土壤酶活性之间的相互关系,寻找人参生长与土壤生物活性之间的变化规律,旨在从生物活性角度分析人参连作障碍成因及为科学指导栽参提供理论依据。分析表明,随着人参生长年限的增加,AWCD值、物种丰富度指数和碳源利用丰富度指数均降低,而优势度度指数升高。土壤脲酶,蔗糖酶活性随栽培年限增加活性降低,而过氧化氢酶活性升高。相关性分析表明,微生物代谢与土壤酶活性关系密切,微生物种类多,奇异度高,微生态系统稳定,土壤酶活性高。因此,保证土壤微生物的多样性,保持土壤酶活性,是提高土壤质量的重要手段,也是解决人参连作障碍的有效途径。     

辽东山区林参复合经营土壤质量评价

林参复合经营是辽东山区典型的林下经济模式之一,而林下参的生长对于环境条件要求很高,其中,土壤质量是制约人参生长的重要因素之一,并对人参产业的健康发展至关重要。目前,阔叶混交林、针阔混交林和蒙古栎纯林是辽东山区广泛分布的林参复合经营林分类型,选择3种林分下立地特征相似、人参种植年限不同的样地,测定土壤物理、化学和生物学因子共25项指标,采用方差分析、主成分分析、相关分析等统计学方法和专家经验法构建了林参复合经营土壤质量评价最小数据集(MDS),并利用模糊数学法对不同样地土壤质量进行了定量评价。结果表明,研究区林参复合经营土壤质量评价最小数据集包括容重、pH、有机质、速效K、全Zn、微生物量N、酸性磷酸酶和细菌共8项指标。此外,土壤质量值由高到低依次为针阔混交林对照样地(0.764)蒙古栎纯林对照样地(0.745)针阔混交林14a人参样地(0.614)蒙古栎纯林20a人参样地(0.385)阔叶混交林12a人参样地(0.361)阔叶混交林对照样地(0.354)阔叶混交林6a人参样地(0.323)阔叶混交林9a人参样地(0.308)阔叶混交林3a人参样地(0.230),说明林分类型特征和人参种植年限对土壤质量会产生不同程度影响。今后,在辽东山区林参复合经营发展过程中,可以该最小数据集为基础,充分考虑土壤质量状况,并适当进行管理,以保障人参健康生长,促进人参产业可持续发展。     

Simultaneous Determination and Analysis of Major Ginsenosides in Wild American Ginseng Grown in Tennessee

Ginsenosides are the major constituent that is responsible for the health effects of American ginseng. The ginsenoside profile of wild American ginseng is ultimately the result of germplasm, climate, geography, vegetation species, water, and soil conditions. This is the first report to address the ginsenoside profile of wild American ginseng grown in Tennessee (TN), the third leading state for production of wild American ginseng. In the present study, ten major ginsenosides in wild American ginseng roots grown in TN, including Rb1, Rb2, Rb3, Rc, Rd, Re, Rf, Rg1, Rg2, and Rg3, were determined simultaneously. The chemotypic differences among TN wild ginseng, cultivated American ginseng, and Asian ginseng were assessed based on the widely used markers of ginsenoside profiling, including the top three ginsenosides, ratios of PPD/PPT, Rg1/Rb1, Rg1/Re, and Rb2/Rc. Our findings showed marked variation in ginsenoside profile for TN wild ginseng populations. Nevertheless, TN wild ginseng has significant higher ginsenoside content and more ginsenoside diversity than the cultivated ginseng. The total ginsenoside content in TN wild ginseng, as well as ginsenosides Rg1 and Re, increases with the age of the roots. Marked chemotypic differences between TN wild ginseng and cultivated American ginseng were observed based on the chemotypic markers. Surprisingly, we found that TN wild ginseng is close to Asian ginseng with regard to these characteristics in chemical composition. This study verified an accessible method to scientifically elucidate the difference in chemical constituents to distinguish wild from the cultivated American ginseng. This work is critical for the ecological and biological assessments of wild American ginseng so as to facilitate long‐term sustainability of the wild population.     

微生物菌剂用于连续移栽人参实验研究

和大多数植物一样,人参存在着难以解决的不能重茬和连作的问题,应用化学方法和采用复合微生物菌剂进行土壤杀菌、土壤复菌等综合性处理试验后,找到了解决问题的关键,介绍应用复合微生物菌剂进行老参地、农田地移栽人参的研究结果。     

栽参对土壤微生物生态及土壤酶活性的影响

我国人参种植历史最久,面积最大,总产量最高。现在,随着国内外市场对人参需求的增加,植参面积还在迅速扩大。但是,人参最忌连作,凡栽种过人参的土地,一般在三、四十年内不能再植参,否则人参大部分乃至全部烂掉。这种土地一般俗称为“老参地”。建国以后,曾对老参地形成和改造进行过许多试验研究,但均未奏效。迄今为止,我国还在沿用     

放线菌制剂对人参生长及根域土壤微生物区系的影响

以小兴安岭地区人参为研究对象,探索放线菌制剂对人参的促生效应及对人参根区、根表土壤微生物区系的影响.结果表明： 经放线菌制剂Streptomyces pactum（Act12）处理后,人参药用部分产量增加,品质改善;叶片诱导酶活性提高,根系活力增强;土壤中细菌、放线菌的数量和比例显著增加,真菌的数量和比例减少.与对照相比,土壤微生物区系结构改变:优势菌荧光假单胞菌、韩国假单胞菌和氧化微杆菌在根区、根表土壤中的数量大幅提高;病原真菌烟色织孢霉在根区土壤中减少,在根表土壤中消失.表明施用放线菌制剂Act12能够改善土壤微生物区系,提高人参植株的抗性和根系活力,增加产量并改善品质.     

植茶年限对土壤微生物群落结构及多样性的影响

为探明植茶年限对土壤微生物群落结构及多样性的影响,以0、20、25、38和48年茶园土壤表层(0～20 cm)、亚表层(20～40 cm)土壤样品为研究对象,采用T-RFLP技术及qPCR方法对土壤细菌(B)、真菌(F)群落进行分析。结果表明: 植茶后土壤理化性质明显改变,随植茶年限的增加土壤有机碳、碱解氮及有效磷含量呈先升高后降低的趋势,表层土壤有机碳和全氮含量均显著高于亚表层土壤。不同植茶年限土壤细菌群落组分存在差异且多样性指数随植茶年限的增加呈下降趋势,而不同植茶年限土壤真菌群落组分差异不明显且多样性指数无显著差异。总体来看,土壤细菌群落对植茶年限的响应比真菌群落敏感。随植茶年限的增加,茶园土壤微生物群落有从F/B较低的“细菌型”向F/B较高的“真菌型”转变的趋势。     

西洋参与人参中人参皂甙含量的比较

采用ＴＬＣ和ＨＰＬＣ方法分析比较西洋参（Ｐａｎａｘ ｑｕｉｎｑｕｅｆｏｌｉｕｍ Ｌ．）、人参（Ｐ．ｇｉｎｓｅｎｇ Ｃ．Ａ．Ｍｅｙ．）及其加工品红参（ｒｅｄ ｇｉｎｓｅｎｇ）,以及不同规格的西洋参中人参皂甙的含量。结果表明,西洋参中人参皂甙总量及人参二醇型皂甙的含量明显高于人参及红参,且含有１种人参及红参中未发现的未知人参皂甙Ｒｘ,但不含人参及红参中含有的Ｒｆ;人参中人参二醇型皂甙的含量高于人参三醇     

人参大片段DNA(100kb)转化灵芝的研究

为探讨人参大片段DNA转化灵芝的可能性,通过电击法将双元细菌人工染色体(BIBAC)载体上的100kb人参大片段DNA转化到灵芝原生质体内。研究发现,在电极间距为4mm,电压强度为240V时,将5μL的人参大片段DNA转化到75μL的灵芝原生质体,在选择培养基上获得了具有再生能力的转化子。根据克隆载体两侧的序列设计两对引物,对转化子进行PCR分析。试验结果表明人参大片段DNA已经转化到灵芝的基因组中。     

Spatial and temporal dynamics of the microbial community structure in the rhizosphere of cluster roots of white lupin (Lupinus albus L.)

White lupin was grown in a quartz sand–soil mix with poorly available Ca phosphate. The plants were harvested on days 21, 35 and 51 and DNA was extracted from the non-cluster roots, the young, mature and senescent cluster roots with adhering soil. Bacterial community structure was examined by PCR-DGGE of 16S rDNA, digitisation of the band patterns and multivariate analyses. In all root zones the bacterial community structure changed with plant age. The communities in the rhizosphere of the non-cluster roots were always different from those of the cluster roots. The bacterial communities of the cluster roots were cluster age and plant age dependent. The differences in bacterial community structure between the cluster root age classes were significant on days 35 and day 51 but not on d 21. A separate experiment, in which root exudates and samples for PCR-DGGE were collected simultaneously, showed that both bacterial and eukaryotic (18S rDNA) community structures change with organic acid exudation. While eukaryotic community structure of the cluster roots was correlated with citric acid exudation, bacterial community structure was correlated with cis-acconitic, citric and malic acid exudation.     

不同恢复年限老参地土壤养分以及酚酸类代谢物含量差异

人工种植西洋参(Panax quinquefolius)具有很高的经济效益, 但连作障碍已成为其产业可持续发展的限制因子。目前对连作障碍成因的研究尚且不足。该研究以收获西洋参后恢复1、10、20年的老参地(分别记为A1、A10、A20)为研究对象, 以未种植过西洋参的土地为对照(CK), 测定和分析土壤养分及酚酸类代谢物的变化, 以期从养分和化感作用的角度解析可能造成西洋参连作障碍的关键因子。通过常规化学性质测定方法和气相色谱质谱联用(GC-MS)的方法测定土壤养分含量, 采用高效液相色谱法(HPLC)测定土壤中的酚酸类代谢物含量。结果显示, 3组收获西洋参后的老参地的土壤pH均显著降低; A1 25种有机态养分(氨基酸类、糖类和糖醇类物质)的含量显著降低, N-乙酰鸟氨酸、5-氨基戊酸、丝氨酸、亮氨酸、甘油和槐糖等的含量均在所有老参地中显著下降, 经过20年轮作后依然不能恢复到对照水平。同时, 与预期相反, 被认为具有化感自毒作用的酚酸类代谢物在收获西洋参后含量也显著下降, 其中, 香豆酸、原儿茶酸、阿魏酸和苯甲酸的含量在A1中显著低于CK, 但经过10年时间轮作后可以恢复到接近对照水平。另外, p-香豆酸和丁香酸在A1、A10、A20的含量均显著低于CK, 即经过20年轮作依然不能恢复到对照水平; 酚酸类代谢物对西洋参生长的积极意义应被重视。相关性分析显示上述有机态养分含量、pH和酚酸类代谢物含量之间大多数呈显著正相关关系, 表明各土壤特性之间存在密切的交互作用。综上所述, 种植西洋参引起的土壤酸化、有机态养分和酚酸类代谢物含量降低及各因子间的协同作用可能是西洋参连作障碍的关键因素。     

Diversity of endophytic bacteria in ginseng and their potential for plant growth promotion

Endophytic bacteria have been found in virtually every plant studied, where they colonize the internal tissues of their host plant and can form a range of different beneficial relationships. The diversity of bacterial endophytes associated with ginseng plants of varying age levels in Korea was investigated. Fifty-one colonies were isolated from the interior of ginseng stems. Although a mixed composition of endophyte communities was recovered from ginseng based on the results of 16S rDNA analysis, bacteria of the genus Bacillus and Staphylococcus dominated in 1-year-old and 4-year-old plants, respectively. Phylogenetic analysis revealed four clusters: Firmicutes, Actinobacteria, α-Proteobacteria, and γ-Proteobacteria, with Firmicutes being predominant. To evaluate the plant growth promoting activities, 18 representative isolates were selected. Amplification of nifH gene confirmed the presence of diazotrophy in only two isolates. Half of the isolates solubilized mineral phosphate. Except four, all the other endophytic isolates produced significant amounts of indole acetic acid in nutrient broth. Iron sequestering siderophore production was detected in seven isolates. Isolates E-I-3 (Bacillus megaterium), E-I-4 (Micrococcus luteus), E-I-8 (B. cereus), and E-I-20 (Lysinibacillus fusiformis) were positive for most of the plant growth promoting traits, indicating their role in growth promotion of ginseng.     

Chemotactic response of Ginseng bacterial soft-rot to Ginseng root exudates

Our purpose was to evaluate chemotactic response of Ginseng bacterial soft-rot to ginseng root exudates. The exudates of plant roots has a significant influence on the population changes of rhizosphere microorganisms and chemotaxis is an important way in which many pathogens sense the signals of host plants and invade the host plants. In this study, with the capillary method, we tested the chemotactic responses of Ginseng bacterial soft-rot for three ginseng roots exudates under four chemotactic parameters (concentration, temperature, pH and time). The results showed that the chemotatic response of the Ginseng bacterial soft-rot for the ginseng roots exudates at the water layer where pH = 7 and the concentration was 0.0125 mg/L reached its peak value under the circumstance that the exudates was cultivated for 60 min at 25 °C. The chemotatic ratios were respectively 124.89% and 89.44%. For the butanol extract layer and the petroleum ether faction at the concentration of 0.125 mg/L and the pH value at 7, the ginseng roots exudatess reached peak values at 25 °C and 30 °C and 60 min and 75 min respectively, and the chemotatic ratios were respectively 139.64% and101.87%, and 115.29% and 81.36%. The three ginseng roots exudates had positive effects for the chemotaxis of the Ginseng soft-rot bacteria, but the effect declined as the concentration increased.     

Biodiversity and distribution of endophytic fungi associated with <Emphasis Type="Italic">Panax quinquefolium</Emphasis> L. cultivated in a forest reserve

Endophytic fungi residing in Panax quinquefolium (American ginseng) have not been well characterized. We collected American ginseng of one-, two-, three- and four-year-old plants cultivated in a forest reserve and identified the endophytic fungal isolates using traditional methods. The colonization frequency and the dominant endophytic fungal species were investigated. Endophytic fungal diversity indices and similarity coefficient were also assessed and all age groups of American ginseng studied were colonized by endophytic fungal assemblages; 134 fungal isolates were assigned to 27 taxa. The infection frequencies varied with the host age and tissue. The dominant endophytic fungi were recorded for each tissue and age of host. The roots of two- and four-year-old American ginseng exhibited the highest and the lowest Shannon-Wiener index respectively. Four-year-old American ginseng had a low similarity coefficient when compared with each of the other three ages classes. The possible role of endophytic fungi in relation to American ginseng cultivation is discussed.