不同探针大小、标记物及反应底物对马铃薯类病毒杂交检测的影响

 灵敏可靠地检测马铃薯纺锤块茎类病毒(Potato spindle tuber viroid,PSTVd)对脱毒种薯的生产具有重要意义。本研究探索了影响核酸杂交检测技术的关键因素。通过基因克隆技术构建了插有PSTVd全长单体、双体及片段的载体。分别以地高辛和同位素为标记物,利用PCR和转录标记技术制备cDNA和RNA探针。比较探针大小、标记物、标记方法、反应底物等对检测灵敏度的影响。结果显示,以地高辛为标记物,利用PCR标记制备的PSTVd双体cDNA探针,在以CDP-Star为底物,通过在柯达X-OMAT BT胶片进行化学发光反应来分析结果的检测灵敏度最高,可以检测到0.05 pg总RNA中的PSTVd,是国外报道检测灵敏度的500倍。利用核酸斑点杂交技术检测PSTVd具有灵敏度高,一次可检测样品数量多等特点,对于大规模PSTVd检测更加方便可行。     

The response of plant species used in agriculture and horticulture to viroid infections

The reactions of the viroids causing cucumber pale fruit (CPFV), chrysanthemum stunt (CSV) and citrus exocortis (CEV) in different plant species and varieties used in agriculture and horticulture were studied. These viroids caused identical symptoms on tomato and potato. The reactions of chrysanthemum Mistletoe to CPFV or CSV were identical, but CEV-infected plants reacted quite differently. CPFV and CEV caused the same type of symptoms on cucumber plants but symptoms of CEV were always weaker.Ten of the tested horticultural crop species appeared to be susceptible to CPFV, of which eight produced symptoms. In the Netherlands, however, CPFV is only described as pathogen on cucumber plants grown in glasshouses. Ten species were susceptible to CSV of which three produced symptoms. In the Netherlands CSV only occurs in chrysanthemum. Nine of the tested crops proved to be susceptible to CEV and seven were sensitive to this viroid which occurs in citric crops of (sub)tropical climate zones.Our results show that the viroids studies form potential pathogens for other crops than cucumber and chrysanthemum which are now affected by two of them.Samenvatting Het viroïde dat de bleke-vruchtenziekte bij komkommer veroorzaakt (CPFV), chrysanthemum stunt viroïde (CSV) en citrus exocortis viroïde (CEV) produceren symptomen op tomaat en aardappel, die niet van elkaar zijn te onderscheiden. De reactie van chrysant Mistletoe op CPFV of CSV is identiek, maar na infectie met CEV reageren de planten volkomen anders. Komkommers ontwikkelen dezelfde symptomen na een besmetting met CPFV en CEV, hoewel laatstgenoemd agens altijd een zwakker ziektebeeld oproept.Tomaat, aardappel, aubergine, chrysant, komkommer, augurk, meloen enBenincasa bleken gevoelig te zijn en paprika en petunia vatbaar voor CPFV, terwijl in Nederland alleen van komkommergewassen bekend is, dat ze aangetast worden door dit pathogeen.Tomaat, aardappel en chrysant waren ook gevoelig voor CSV, vatbaar voor dit viroïde waren paprika, petunia,Nicandra, andijvie, komkommer, augurk enBenincasa cerifera. In ons land kennen we stunt alleen in de chrysantenteelt. Negen gewassen bleken vatbaar te zijn voor CEV, en zeven waren gevoelig voor dit viroïde, dat alleen in (sub)tropische klimaatzones voorkomt.Vierentwintig cultuurgewassen bleken onvatbaar te zijn voor alle drie viroïden. De resultaten van dit onderzoek wijzen er op dat CPFV, CSV en CEV nauw verwant zijn, maar niet identiek.     

The distribution of potato spindle tuber viroid in potato plants and tubers1

Potato spindle tuber viroid (PSTV) in potato plants was investigated by ‘return’ gel electrophoresis. The experiments were carried out under quarantine conditions in the greenhouse with primarily and secondarily infected plants. The PSTV content in different plant parts was estimated by the intensity of the viroid band in polyacrylamide gel. The results showed a decrease of viroid content from the upper to the lower parts of the plant. In both primarily and secondarily infected plants, PSTV was reliably detected in the top leaves, but less so in the lower leaves. In four out of ten secondarily infected plants, PSTV was found in the roots. In dormant tubers, the bands were more intense with samples obtained from the rose end and the heel than from those obtained from the medullary tissue. With one exception, all 64 tubers from 26 primarily infected plants were infected with PSTV.     

4个马铃薯品种感染马铃薯纺锤块茎类病毒（PSTVd）的症状

马铃薯纺锤块茎类病毒在中国及世界许多国家都是检疫性病害, 该病害可大幅度降低马铃薯产量和品质。根据植株及块茎的症状, 汰除感病的马铃薯种薯, 是控制PSTVd传播和危害的重要手段。本研究利用分子克隆及测序技术, 分离并鉴定了PSTVd的分离物351-17, 利用PSTVd田间接种技术, 分别接种了4个黑龙江省常用的马铃薯品种‘夏坡地’、‘克新18’、‘荷兰15’和‘尤金’, 对其侵染后第一代的株高、叶片、块茎外观和单株产量进行了调查, 结果表明：4个供试马铃薯品种感染PSTVd后均表现出了一定的症状, 其中‘克新18’在株高、叶片和块茎的症状方面最为明显, 单株产量下降显著, 表明其对PSTVd非常敏感。     

多重RT-PCR同时检测6种马铃薯病毒及1种类病毒

病毒病是限制我国马铃薯生产的重要因子之一。本研究通过引物设计和体系优化建立了一套多重RT-PCR检测方法,可以在一个反应体系中同时检测6种马铃薯病毒和1种马铃薯类病毒以及1个马铃薯内参基因,包括马铃薯Y病毒(potato virus Y, PVY)、马铃薯M病毒(potato virus M, PVM)、马铃薯S病毒(potato virus S, PVS)、马铃薯X病毒(potato virus X, PVX)、马铃薯A病毒(potato virus A, PVA)、马铃薯卷叶病毒(potato leaf curl virus, PLRV)、马铃薯纺锤块茎类病毒(potato spindle tuber viroid, PSTVd)和细胞色素c氧化酶亚基Ⅰ(cytochrome c oxidase subunitⅠ,CoxⅠ)基因的特异片段,产物大小依次为181、226、275、565、630、681、359、500 bp,符合理论预期。采用建立的方法以相应病毒和类病毒的质粒作模板进行检测,其检测灵敏度在1.6×10^-3～1.8×10^-1 ...     

Transmission of potato spindle tuber viroid by aphids

Aulacorthum solani (Kaltenbach),Macrosiphum euphorbiae (Thomas) andMyzus persicae (Sulzer) were used for transmission experiments in laboratory and glasshouse. As inoculum served PSTV-containing tomato foliage and artificial diets containing purified PSTV. It is concluded thatM. euphorbiae can transmit PSTV in a non-persistent way.Samenvatting Aardappelspindelknolviroïde (PSTV) wordt tot een vrij nieuwe groep van pathogenen, de viroïden, gerekend. De ziekte, die er door wordt veroorzaakt, wordt sedert 1923 waargenomen in Noord-Amerika en is meer recentelijk ook aangetroffen in Afrika, Australië, Sowjet-Unie en Zuid-Amerika maar tot dusver niet in West-Europa.PSTV kan gemakkelijk mechanisch worden overgebracht. De overdracht door bladluizen was nog niet betrouwbaar vastgesteld. De bladluissoortenMyzus persicae (Sulzer),Aulacorthum solani (Kaltenbach) enMacrosiphum euphorbiae (Thomas) zijn daarom in overdrachtsproeven betrokken, waarbij als inoculum met PSTV geïnfecteerd blad en een door een membraan afgesloten gezuiverde PSTV-bevattende suspensie is gebruikt. Het laatstgenoemde type inoculum is gebruikt, om een mogelijke mechanische overdracht bij het overzetten van bladluizen van de viroïdebron naar de toetsplant tomaat cv. Sheyenne uit te sluiten. Er is vastgesteld, datM. euphorbiae PSTV op non-persistente wijze kan overbrengen.     

Simplified application of return gel electrophoresis for the routine detection of potato spindle tuber viroid

The return gel electrophoresis method for the detection of potato spindle tuber viroid has been modified to simplify its use for large-scale testing. The quantities of dissolvents for the extraction of nucleic acids were reduced and adapted to the use of disposable plastic tubes. The vertical electrophoresis system was replaced by a horizontal one, which was easier to handle. Migration distance for the separation of PSTV bands was shortened, saving time for the electrophoretic run. A single-buffer system was used. The detection limit was estimated as 0.3 ng per slot. It was possible to detect both mild and severe strains of PSTV.     

Transmission by aphids of potato spindle tuber viroid encapsidated by potato leafroll luteovirus particles

Potato spindle tuber viroid (PSTVd) was transmitted by Myzus persicae to Physalis floridana from P. floridana plants that also were infected with potato leafroll luteovirus (PLRV), whereas it was not transmitted by aphids from plants infected with PSTVd alone. Dot-blot hybridisation was used to detect PSTVd. The results indicate that PLRV can assist PSTVd in its transmission by M. persicae. Doubly infected, aphid-inoculated P. floridana plants from the previous experiment were used as the source plants in aphid transmission tests to the tomato cv. Rutgers, P. floridana and Datura stramonium. PSTVd was detected in 17 of 30 plants of tomato. The viroid was not detected by dot-blotting in any plant of P. floridana and D. stramonium in this experiment, but it was recovered from some plants by sap inoculation of the Rutgers plants. Treatment with RNase A of PLRV preparations purified from doubly infected plants indicated that PSTVd was encapsidated by PLRV particles.     

Genetic diversity and pathogenicity of potato spindle tuber viroid and chrysanthemum stunt viroid isolates in Russia

European Journal of Plant Pathology - To investigate the current status of viroid infection in potato fields in Russia, potato spindle tuber viroid (PSTVd) and chrysanthemum stunt viroid (CSVd)...     

甘薯褪绿矮化病毒西非株系实时荧光定量PCR检测方法的建立及应用

 依据GenBank中登录的甘薯褪绿矮化病毒(Sweet potato chlorotic stunt virus,SPCSV)西非株系(WA)的核苷酸序列,分别设计两对特异性引物和一条TaqMan探针。以SPCSV-WA外壳蛋白(cp)基因的重组质粒为阳性标准质粒绘制标准曲线,通过优化反应体系和反应条件,建立了SPCSV-WA的实时荧光定量PCR检测方法。试验结果表明,该方法只能检测到目的病毒,标准曲线的斜率和相关系数分别为-3.239和1,扩增效率为103.568%。最低可检测到约3.31 copies/μL的阳性质粒,灵敏度比常规PCR高1 000倍。本研究建立的SPCSV实时荧光定量PCR方法可用于田间样品的检测,为SPCSV的早期预警和流行学研究提供了技术手段。     

灌溉量对马铃薯生理特性及 块茎产量品质的影响

以 “大西洋”马铃薯品种为试验材料,在甘肃省中部沿黄高扬程灌区的白银市景泰县条山农场马铃薯种植基地设置小区试验,在各发育阶段分别设置5个水分梯度（原灌溉量的50%、75%、100%、125%和150%,其中原灌溉量,即100%灌溉量,为当地常用的、维持试验地25.8%左右土壤含水率的灌溉量）,探讨马铃薯各发育阶段灌溉量对其生理特性及块茎产量、品质的影响。结果表明,在马铃薯的各发育阶段,随着灌溉量的增加马铃薯叶片净光合速率 (P_n)和蒸腾速率(T_r)分别先增加到最大值30.19 μmol·m^-2·s^-1、12.50 mmol·m^-2·s^-1,而后减小。当灌溉量为50%和150%时叶片P_n较100%灌溉量分别降低35.06%和19.59%。在不同发育阶段马铃薯叶片的生理活性对灌溉量表现出敏感响应。幼苗期75%灌溉量马铃薯叶片叶绿素含量、P_n和水分利用效率（WUE）分别为0.86 mg·g^-1、21.88 μmol·m^-2·s^-1、2.29 μmol·mmol^-1,较100%灌溉量分别提高了14.15%、0.31%、3.80%,而MDA含量、SOD活性及CAT活性最低,较100%灌溉量分别降低了8.40%、11.69%、8.23%;发棵期100%灌溉量处理下P_n和WUE最高,较50%灌溉量处理分别提高了53.99%、24.85%,此时脯氨酸含量、可溶性糖含量、POD活性最低,分别为89.06 μg·g^-1、143.45 μmol·g^-1、438.14 U·g^-1,同时超氧阴离子产生速率较50%灌溉量也降低了26.07%;结薯期125%灌溉量处理的P_n和WUE最高,较100%灌溉量分别提高了19.16%、6.76%,而SOD、POD活性较100%灌溉量分别降低了0.93%、6.23%;成熟期75%灌溉量马铃薯叶片P_n最高,较其余各灌溉量处理的P_n分别提高了38.64%、13.81%、14.16%、21.27%,而MDA含量、超氧阴离子产生速率分别为21.107 μmol·g^-1、6.07 nmol·min^-1·g^-1,较100%灌溉量分别降低了19.35%、2.26%。同一生育期低灌溉量（50%灌溉量）块茎中淀粉含量较低。在幼苗期75%灌溉量处理使得块茎的可溶性蛋白含量较100%灌溉量处理提高6.20%。发棵期100%灌溉量块茎中淀粉含量达到最高（15.14%）,而结薯期125%灌溉量处理的块茎可溶性蛋白含量处于最高水平,较100%灌溉量下提高4.61%。成熟期75%灌溉量块茎淀粉含量处于最高水平,较100%灌溉量提高12.77%。结合块茎的总鲜重、商品薯率、淀粉含量、维生素C含量、可溶性蛋白含量、还原糖含量等指标,可知在幼苗期、发棵期、结薯期、成熟期灌溉量分别为原灌溉量的75%、100%、125%、75%时马铃薯块茎品质最优而且产量最高,分别为654.30、650.60、773.00、703.53 g·株^-1。     

Occurrence and molecular variability of Potato spindle tuber viroid and Tomato apical stunt viroid in ornamental plants in Croatia

Viroids of the genus Pospiviroid are able to induce diseases in a wide range of host plants including important crop species. Although occasional disease outbreaks of Potato spindle tuber viroid (PSTVd) and closely related pospiviroids have been reported in potato and tomato, recent studies found an increase in number of latent infections in ornamental solanaceous species. In order to verify the presence of PSTVd and other pospiviroids in Croatia, a survey was conducted between 2009 and 2012. A total of 182 samples belonging to five ornamental species and two solanaceous crops were analyzed. Eight plants belonging to two different species (Solanum jasminodes and Lycianthes rantonnetii) were found infected by PSTVd and, in addition, one S. jasminoides plant infected by Tomato apical stunt viroid (TASVd). Viroid infection was confirmed by mechanical inoculation on tomato plants to observe symptom expression. Molecular characterization of the isolates was done and complete viroid sequences were submitted to the GenBank. This is the first evidence of the presence of PSTVd and TASVd and their variability in Croatia.     

取食马铃薯块茎和叶片的马铃薯块茎蛾肠道内可培养细菌的多样性

为明确取食马铃薯块茎和叶片的马铃薯块茎蛾Phthorimaea operculella幼虫肠道可培养细菌种类组成的多样性及其差异,分别对取食马铃薯块茎和叶片的马铃薯块茎蛾4龄幼虫的肠道细菌进行分离培养,根据菌落形态和生理生化特征及16S rDNA序列分析对菌株进行分类鉴定。结果表明,从取食马铃薯块茎的马铃薯块茎蛾肠道内共分离获得细菌有4门8科10属15种,其中褪色沙雷氏菌Serratia marcescen和乙酸钙不动杆菌Acinetobacter calcoaceticus为优势种,其相对多度分别为17.20%和16.06%。从取食马铃薯叶片的马铃薯块茎蛾肠道内共分离获得细菌有4门10科10属16种,其中琥珀葡萄球菌Staphylococcus succinus和乙酸钙不动杆菌为优势种,其相对多度分别为11.86%和15.07%。取食马铃薯块茎和叶片的马铃薯块茎蛾肠道内相同的可培养细菌有4属5种,分别为褪色沙雷氏菌、深红沙雷氏菌Serratia rubidaea、阿氏肠杆菌Enterobacter asburiae、乙酸钙不动杆菌和琥珀葡萄球菌。表明取食马铃薯块茎和叶片的马铃薯块茎蛾...     

马铃薯块茎病原真菌拮抗菌株筛选及优良拮抗菌株鉴定

从马铃薯块茎上分离内生细菌,以块茎病原真菌为指示菌,采用平板对峙法筛选拮抗菌株,并利用形态学特征和16S rDNA基因序列分析对优良拮抗菌株进行鉴定。结果表明,从定西马铃薯块茎中共分离得到72株内生细菌,其中19株对5种块茎病原真菌的抑菌率均高于51.87%,特别是内生细菌6-5和5-6分别对马铃薯炭疽病菌Colletotrichum coccodes和马铃薯黑痣病菌Rhizoctonia solani的抑菌率达85.82%和72.18%,且具有固氮和产吲哚乙酸(IAA)功能,其产IAA量分别为3.37 mg/L和19.25 mg/L;根据形态特征及16S rDNA基因序列将菌株5-6鉴定为萎缩芽胞杆菌Bacillus atrophaeus,菌株6-5鉴定为贝莱斯芽胞杆菌Bacillus velezensis。     

啤酒花矮化类病毒实时荧光定量RT-PCR检测方法的建立与应用

为建立一种快速、灵敏、特异的定量检测啤酒花矮化类病毒(Hop stunt viroid,HSVd)的实时荧光定量RT-PCR (RT-qPCR)方法,设计了2对引物及特异探针,体外转录制备了RNA标准品,绘制标准曲线,并对该方法的特异性、灵敏度和重复性进行评估.建立的定量标准曲线Ct值与模板拷贝数对数之间呈良好的线性关系,相关系数R2为0.9988,扩增效率为95％;该方法的特异性好,与啤酒花潜隐类病毒(HLVd)、葡萄黄斑类病毒1(GYSVd-1)、葡萄黄斑类病毒2(GYSVd-2)和桃潜隐花叶类病毒(PLMVd)均无交叉反应;灵敏度为1.0×102拷贝/μL,比普通RT-PCR高10倍;试验内及试验间重复性试验的变异系数均小于3％.研究表明该方法适用于实际样品中HSVd的快速定量检测.     

广东省马铃薯块茎软腐病病原菌鉴定

为明确广东省惠州市马铃薯块茎软腐病的病原菌,采用常规病组织分离法获得4株菌株,通过生理生化特征和分子生物学特性对其进行鉴定,并测定了该病原菌对20个马铃薯品种的致病力。结果显示,该病原菌为革兰氏阳性菌,在LB平板上菌落有透明和不透明2种形态;除葡萄糖发酵阳性、对链霉素和青霉素具有抗性等特征不同外,BP-hd-1、BP-hd-2、BP-hd-3和BP-hd-4菌株与短小芽胞杆菌Bacillus pumilus菌株的其它生理生化特征均相同。系统进化分析结果表明,4株菌株的16S rDNA序列分别与短小芽胞杆菌AI-Khrj18(KY123871)、ML270(KC692158)、NCTC10337(LT906438)和ARD21(KX023236)的相似性为100.0%;4株菌株的gyrB基因序列均与短小芽胞杆菌AUEC29菌株(HM585095)的gyrB基因相似性最高,为99.7%～99.8%。生理生化特征及分子生物学鉴定结果表明,引起广东省马铃薯块茎软腐病的病原菌为短小芽胞杆菌。致病力测定结果显示,接种菌株BP-1后20个马铃薯品种的发病率均为100.0%,表明该病原菌对马铃薯有强致病力。