三七根腐病株根际土壤真菌群落组成与碳源利用特征研究

根腐病是药用植物三七Panax notoginseng的多发性病害,而土壤真菌群落组成与根腐病的发生存在一定的联系。利用末端限制性酶切片段长度多态性分析和Biolog技术研究了云南文山三七种植园健康和发病三七植株根际土壤真菌群落结构和碳源利用特征,结果表明健康和发病植株的根际土壤真菌多样性和代谢活性差异不显著,但是真菌群落组成存在差异。健康和发病植株根际土壤都以小茎点霉Phoma exigua及镰孢菌属Fusarium真菌为主要种群,而健康植株根际土壤中火丝菌属Pyronema和被孢霉属Mortierella等真菌类群的相对丰度显著高于发病植株根际土壤,尖孢镰孢菌F.ox‐ysporum和刺座霉属的Volutella colletotrichoides的相对丰度则显著低于发病植株。     

深黄被孢霉△5-脱饱和酶基因在花生四烯酸合成中的作用

【目的】研究△5-脱饱和酶基因的mRNA表达量与花生四烯酸产量之间的关系。【方法】实验利用荧光定量PCR方法检测了△5-脱饱和酶在五株深黄被孢霉的同一培养时间及菌株YZ-124在不同发酵阶段的mRNA表达水平,同时利用气相色谱仪测定其花生四烯酸含量。【结果】结果表明:不同菌株的△5-脱饱和酶基因的mRNA表达水平不同,原始菌株As3.3410最低,诱变菌株YZ-124最高;深黄被孢霉YZ-124不同发酵阶段的△5-脱饱和酶基因mRNA表达量随菌龄的增加逐渐增加。【结论】结合ARA的得率显示,△5-脱饱和酶基因mRNA表达量与培养物油脂中ARA含量呈一定的正相关关系。     

小麦根腐病菌索氏平脐蠕孢SYBR Green I实时荧光定量PCR检测技术研究

由索氏平脐蠕孢Bipolaris sorokiniana引起的小麦根腐病,常和其他土传真菌病害混合发生,传统的症状鉴别方法很难区分,导致病害防控难度增加。为建立病菌实时荧光定量检测体系,根据ITS序列设计引物,筛选出1对特异性引物BS‐F/R,扩增片段大小为280bp。以菌丝DNA为标准品构建实时荧光定量标准曲线,并对其灵敏度、特异性、可重复性进行评价。结果表明,建立的实时荧光定量PCR检测方法速度快,灵敏度高,特异性强,重复性好。构建的荧光定量PCR标准曲线循环阈值与模板浓度呈良好的线性关系,溶解曲线的吸收峰单一,扩增效率良好。利用该定量检测体系,可以检测出田间小麦样品中52.8fg/μL的病菌DNA。     

引起青稞和燕麦鞘腐病的无性型菌物新属种——禾生指葡孢霉

引起甘肃及邻省青稞和燕麦鞘腐病的病原菌为无性型真菌anamorphic fungi中一未描述的丛梗丝孢菌moniliaceous hyphomycete。该菌分生孢子梗无色,散生,直立或匍匐;交互、叉状、聚伞状或帚状分枝。产孢细胞簇生于分生孢子梗末级分枝顶端成指状或掌状,顶部常具齿状着孢点,全壁芽生式产孢。分生孢子无色、单胞,罕有一个隔膜,近球形、卵圆形、倒梨形、纺锤形、葫芦形或不规则形,表面具网状脊或疣突,在孢梗顶端常聚集成葡萄状。分生孢子群体白色,随培养物老化,渐变为淡黄色。本菌与葡萄孢属Botrytis及其他Botrytis-like形态相似属明显不同,故建立指葡孢霉属Dactylobotrys(新属);模式种为禾生指葡孢霉Dactylobotrys graminicola(新种)。模式标本保存在中国科学院菌物标本馆(HMAS 245110,主模式)和甘肃省农业科学院植物保护研究所植物病害标本室(GAPP 1131,等模式)。     

临床皮炎外瓶霉荧光定量PCR检测方法的建立

目的 建立基于TaqMan探针技术的皮炎外瓶霉荧光定量PCR检测方法.方法 通过对皮炎外瓶霉ITS区域基因组序列（GenBank：JN675373.1）进行分析,设计合成特异性引物和荧光标记探针,优化荧光定量PCR反应条件.以临床标本中分离的皮炎外瓶霉为阳性菌株,及其他种类真菌和细菌作为阴性对照菌株,从特异性、敏感性及重复性方面对该方法检测效果进行评价.结果 该研究设计的引物和探针能扩增皮炎外瓶霉特异性序列.临床分离得到的皮炎外瓶霉在反应中有明显扩增曲线,而甄氏外瓶霉、棘状外瓶霉、烟曲霉、白色念珠菌、新生隐球菌、马内菲青霉等20株菌在CT值≤38范围内均未有扩增;利用基因重组构建的标准品完成了标准曲线的绘制,在1.0×103～1.0×107拷贝数（Cp）内具有良好的线性关系（R2=1.000）,最低可检出量为10 Cp/μL.结论 成功建立了荧光定量PCR检测皮炎外瓶霉方法,该法特异度强、敏感度高、重复性好,将有助于临床皮炎外瓶霉感染的早期诊断和针对性治疗.     

华南热带水库拟柱孢藻产毒基因型的低优势及影响因子分析——以珠海市水库为例

研究以珠海市20座水库两次(2018年和2022年)调查数据为基础,分析华南热带地区产毒拟柱孢藻的发生规律和拟柱孢藻毒素(CYN)的健康风险。基于rpoC1和cyrJ基因的荧光定量PCR分析发现,在两次采样的40个样品中均检到拟柱孢藻,在其中的33个样品中检到产毒拟柱孢藻。种群内产毒基因型的比例在0.046%—38.66%,表明产毒株广泛存在但不占优势。与2018年相比, 20座水库的总拟柱孢藻丰度均值在2022年增加近10倍,但两年的产毒拟柱孢藻丰度无显著差异;产毒基因型的比例明显下降,比例均值和最大值分别从2018年的14.86%和38.66%降至2022年的4.17%和17.24%; CYN浓度与产毒细胞比例具有类似的变化趋势,平均浓度也从0.56降至0.19μg/L。线性混合效应模型分析表明,非产毒基因型丰度随无机磷的升高及硝氮的下降而显著增加,而产毒拟柱孢藻比例随水温升高而增加。     

木糖葡萄球菌实时荧光定量PCR检测方法的建立及其应用

目的 本研究拟建立一种灵敏快速的实时荧光定量PCR(real-time quantitative PCR, qPCR)方法,用于检测大、小鼠木糖葡萄球菌(Staphylococcus xylosus,S.xylosus)。方法 本研究选择特异性gehM基因片段作为靶标合成了一套引物,建立了木糖葡萄球菌检测的qPCR方法。对木糖葡萄球菌标准菌株和其他非目标菌进行特异性分析。将木糖葡萄球菌的DNA进行10倍稀释测定其灵敏度。用送检的样本进行了临床应用并测序验证,同时与培养法进行比较。结果 仅木糖葡萄球菌出现特异性扩增曲线,而其他非目标菌未出现,表明设计的引物对木糖葡萄球菌具有特异性,灵敏度为100 fg/μL,组内和组间重复性均小于3%。共检测60份临床样品,有5份样品扩增曲线为典型的S曲线,将该qPCR产物克隆测序并进行同源性比对,该序列与木糖葡萄球菌的同源性为99.63%,表明该样本木糖葡萄球菌核酸阳性,所检测样本阳性率为8.3%,而培养法的阳性率为6.7%,qPCR方法阳性检出率比培养法略高。结论 建立的木糖葡萄球菌qPCR方法,具有快速、灵敏度高、特异性强和重复性好的优点,可用于实...     

实时荧光定量PCR及其在传染性疾病检测中的应用

实时荧光定量PCR技术被广泛应用于实验研究、临床检测中。与普通的PCR相比,实时荧光定量PCR技术具有特异性强、灵敏度高、重复性好、定量准确、速度快、全封闭反应等优点。我们综述了实时荧光定量PCR技术的原理、定量方法,及其在传染性疾病检测研究中的应用。     

实时定量PCR及其在轮状病毒检测中的应用

实时定量PCR(Real-time polymerase chain reaction/quantitative Real-time polymerase chain reaction,Real-time PCR/qPCR)就是在PCR扩增过程中,通过荧光信号对PCR进程进行实时监测。它具有特异性强、灵敏度高、定量准确和快速等优点,在生物医学领域中得到广泛的应用。对实时定量PCR技术的原理和类型,实时定量PCR技术在生物医学领域的应用,尤其在轮状病毒诊断、检测及疫苗研究中的应用及其未来前景进行了综述。     

三七连作根际土壤微生物区系的16S rRNA系统遗传多样性

【目的】针对三七连作土传病害的生物防治技术研究,探讨其根际土壤微生物多样性与根腐病害的关联性。【方法】采集三七连作6年的健康植株与根腐病植株根际土壤,采用多种培养基进行分离、纯化,并对分离菌株提取DNA,用其16S rRNA序列的通用引物进行PCR扩增,进行Blast同源及其系统进化树分析。【结果】从采集样品中分离出的菌株分布于细菌域(Bacteria)中的4个门(Phyla)共40个属(Genera),其中从健康植株组土壤中培养出179株菌,分布于30个属,以伯克氏菌属(Burkholderia)、节杆菌属(Arthrobacter)、链霉菌属(Streptomyces)及芽孢杆菌属(Bacillus)为优势菌群;根腐病株组土样共培养出117株菌,分布于29个属,以罗尔斯顿菌属(Ralstonia——病原菌)、单胞菌属(Sphingomonas)、寡养单胞菌属(Stenotrophomonas)等为优势菌群;其中黄杆菌属(Flavobacterium)、肠杆菌属(Enterobacter)的分离菌株仅从病株组土壤中分离到;发现其可能新种5株;健康组与根腐病组土壤EC值及各元素离子浓度差异明显(P<0.05),健康组土壤中的优势微生物种群分布与土样中的EC值、NO-3、SO2-4、CO2-3、K+及全盐量呈负相关关系(显著度P<0.01)。【结论】研究表明,诱导三七连作土传病害的发生,除病原菌作用影响外,还与土壤理化性质和土壤微生物的种群结构及其优势微生物的种群比例具有密切的关联性,特别是土壤中有益微生物(Burkholderia,Bacillus,Streptomyces等)的丰度,对评价三七土壤健康状况及病害防治预报具有重要的指导意义。     

云南文山健康三七种植年限对根际微生物群落的影响

【背景】三七的连作障碍与微生物区系失衡有一定关联,了解种植年限对三七微生物区系的影响,判断根际微生物区系是否失衡,对三七连作障碍的防治具有重要意义。【目的】探究文山不同种植年限健康三七根际土壤细菌和真菌群落结构、多样性差异,以及与种植年限之间的关联,为后续缓解三七连作障碍提供理论依据。【方法】采集一年生、二年生和三年生健康三七根际土壤,采用16S rRNA基因和ITS序列进行高通量测序。对测序结果进行分析,比较一年生、二年生和三年生健康三七根际土壤细菌和真菌的多样性和群落分布规律及与种植年限的关联。【结果】一年生健康三七根际细菌和真菌群落的α指数均显著高于二年生和三年生。通过β多样性分析发现,一年生健康三七根际土壤中真菌群落结构与其他年限的真菌群落结构有较大差异。相关性分析结果表明,真菌的均匀度和香农指数与种植年限呈现显著的负相关性,其余指数不显著相关。三七根际丰富度前三的门和属,除Acidobacteria和Aquicella丰富度随着三七种植年限的增加而显著增加外,其余群落丰度都与种植年限呈负相关。细菌中Bacteroidetes、Proteobacteria和真菌中Ascomycota、Basidiomycota、Chytridiomycota的丰富度随着种植年限增加而降低。在属水平上,细菌中Opitutus和真菌中Mortierella、Clitopilus、Pholiota的丰富度随着三七种植年限的增加而降低。在二年生三七根际土壤中病原真菌Alternaria、Cylindrocarpon、Fusarium和Pestalotiopsis的丰富度低于一年生和三年生,而有益真菌Mucor和Bacillus的丰富度却呈相反趋势。同时,有益细菌Flavobacterium和有益真菌Myxocephala、Aspergillus的丰富度随着种植年限的增加呈下降趋势。【结论】三七种植年限的增加,会引起三七根际微生物区系失衡,增加根际病原真菌属的丰度,同时降低一些有益真菌属的丰度。     

Fungicidal Activity of Essential Oils from Cinnamomum cassia against the Pathogenic Fungi of Panax notoginseng Diseases

The frequent disease of Panax notoginseng caused by the pathogenic fungi in field cultivation has become the major threaten to the sustainable development of it. The present study was conducted to find natural agent with potential inhibition against pathogen. Therefore, the inhibitory effects of Cinnamomum cassia (L.) J.Presl essential oils (EOs) against P. notoginseng associated pathogenic fungi were conducted both in vitro and in vivo experiments. The results of the Oxford cup test revealed that C. cassia dry bark EO (50 mg/mL) had significant inhibitory activity on the growth of all tested fungi, and the growth of various pathogens was completely inhibited, except for that of Fusarium solani. Therefore, the constituents of C. cassia EOs were analyzed by GC/MS, and the research demonstrated that the main constituents of C. cassia dry bark EO were trans‐cinnamaldehyde (75.65 %), (E)‐2‐methoxycinnamaldehyde (6.08 %), cinnamaldehyde (3.47 %) and cinnamyl acetate (1.02 %). The MIC results showed that C. cassia dry bark EO and the main compounds had good antifungal effect on the tested strains, and the inhibitory effect was similar to that of hymexazol (chemical pesticide). By analyzing the value of the fraction inhibitory concentration index (FICI), additive effects, irrelevant effects and synergistic effects were observed after the mixture of hymexazol against various pathogens. Moreover, in vivo model showed that C. cassia dry bark EO could reduce the occurrence of anthrax in P. notoginseng. To widen the resources of C. cassia available, the compositions of both C. cassia fresh bark and leaf EOs were also tested and many common compositions existed among them. Taken together, it was concluded that C. cassia EO had the potential use in the field to reduce the pathogenic disease.     

三七生物资源研究与利用进展

三七为五加科人参属植物,因其在抗血栓、抗高血压、保肝、活血化瘀等方面具有显著疗效而闻名中外,是我国独具特色的生物资源,具有十分广阔的产业化前景。本文从三七的植物化学与药理学、基因组学研究和遗传改良等方面综述了目前的研究现状,分析了三七生物资源利用过程中存在的问题,浅谈了未来的研究与利用展望。     

Control of pathogenic fungi on Panax notoginseng by volatile oils from the food ingredients Allium sativum and Foeniculum vulgare

To screen natural drugs with strong inhibitory effects against pathogenic fungi related to P. notoginseng, the antifungal activities of garlic and fennel EOs were studied by targeting P. notoginseng disease-associated fungi, and the possible action mechanisms of garlic and fennel EOs as plant fungicides were preliminarily discussed. At present, the antifungal mechanism of EOs has not been fully established. Therefore, understanding the antifungal mechanism of plant EOs is helpful to address P. notoginseng diseases continuous cropping disease-related obstacles and other agricultural cultivation problems. First, the Oxford cup method and chessboard were used to confirm that the EOs and oxamyl had a significant inhibitory effect on the growth of Fusarium oxysporum. F. oxysporum is the main pathogen causing root rot of P. notoginseng and the preliminary study on the antifungal mechanisms of the EOs against F. oxysporum showed that the inhibition of EOs mainly affects cell membrane permeability and cell processes and affects the enzyme activities of micro-organism, to achieve antifungal effects. Finally, an in vivo model verified that both two EOs could significantly inhibit the occurrence of root rot caused by F. oxysporum.     

正交试验法优选三七茎叶中总黄酮的提取工艺

目的：从三七茎叶中提取总黄酮。方法：采用正交试验法研究三七茎叶总黄酮的提取工艺,考察了浸提用水的pH值、浸提温度、浸提时间、浸提次数、液料比五个因素对三七茎叶总黄酮提取率的影响。结果：确立了三七茎叶总黄酮最佳提取条件为：用pH10的稀碱水作溶剂,液料比30：1,浸提温度为80℃,回流提取2次,提取时间为每次40min。     

Determination of the Heavy Metal Levels in Panax notoginseng and the Implications for Human Health Risk Assessment

ABSTRACT

High levels of heavy metals in Panax notoginseng (Sanchi), a valued traditional Chinese medicine, have drawn increasing concern regarding the safe usage of Sanchi preparations. Here, we measured the concentrations of six heavy metals in Sanchi samples from 20 major plantations, investigated the pharmaceutical processes and usages of Sanchi preparations, and assessed the associated potential health risks to consumers. The average concentrations of chromium (Cr), copper (Cu), nickel (Ni), zinc (Zn), lead (Pb), and arsenic (As) in the Sanchi samples were 2.7, 3.7, 6.2, 22.1, 2.0, and 1.4 mg/kg, respectively. The hazard quotients (HQs) for these six single metals and the hazard index (HI) of these metals’ combination were all far less than 1, indicating the absence of a non-carcinogenic health hazard to consumers. The carcinogenic risk of As was 2.1 × 10^?6, which is higher than the allowable level suggested by the U.S. Environmental Protection Agency but less than the level suggested by the World Health Organization (WHO). The probabilities of consumers’ exposure due to daily medicine consumption exceeding the allowable daily intakes from medicine (ADIs_drug, 1% of the ADI) suggested by the WHO were 0.0%, 0.1%, 0.1%, 0.0%, 1.6%, and 27.3% for Cr, Ni, Cu, Zn, Pb, and As, respectively.