马铃薯黑胫病菌的分离、纯化及PCR检测

由Pectobacterium atroseptica引起的马铃薯黑胫病是影响马铃薯生产的重要种薯传播细菌病害,从种薯发芽到生长后期均可发病,严重影响马铃薯的产量和品质。为构建马铃薯黑胫病菌的分离、鉴定技术体系,从广东省惠州市惠东县大水坑马铃薯基地采集了疑似感染马铃薯黑胫病植株,对其进行病原菌分离和纯化,得到7株病菌,利用引物Eca 1 g/Eca 2 g对其进行PCR扩增、将PCR产物测序并将所测定序列递交Gen Bank数据库,使用Blast软件进行比对确定目标菌株。结果表明,其中一株菌株能扩增出长度大约688 bp的特异性条带,其DNA序列符合Eca 3762序列,鉴定为马铃薯黑胫病菌。此方法可以成功地从发病植株中分离出黑胫病菌,成本较低廉,为广东省建立马铃薯种薯质量检测和控制体系提供技术支撑。     

采用DNA条形码技术检测马铃薯4种细菌病害

马铃薯易受到多种细菌病害的侵染,特别是检疫性病害和土传性病害,对马铃薯种薯进行全面的细菌病害检测势在必行。将检测检疫性细菌病害的DNA条形码技术应用在马铃薯4种细菌性病害(环腐病、青枯病、疮痂病和黑胫病)的检测中,探讨该项技术的应用可行性。采用已知菌株以明确该项技术的应用效果,结果表明,该项DNA条形码技术检测马铃薯环腐病和疮痂病的结果良好;检测青枯病可以确定到属;检测黑胫病时配合特异性基因,可获得良好的检测结果。该DNA条形码技术是标准性操作规程与测序技术相结合的一种方法,检测结果的准确性高,同时可以在大规模样品的检测工作中缩减工作量,提高检测效率。     

马铃薯黑胫病菌检测技术

马铃薯黑胫病是国内外马铃薯产区发生比较普遍的一种细菌性病害,这种病害造成马铃薯块茎损失可高达30%～50%,严重地影响了马铃薯的产量和质量。文章就近几年国内外对该细菌的检测技术,包括症状识别、细菌分离、酶联免疫吸附法、菌落免疫荧光染色法、AFLP指纹识别技术、荧光实时定量PCR检测技术、PCR-RFLP检测技术、T-RFLP检测技术的研究进展做了阐述。     

马铃薯黑胫病的发生与防治

马铃薯黑胫病是一种重要的马铃薯细菌性病害 ,是以茎基部变黑的症状而命名的 ,因此又称“黑脚病”、“黑腿子病”。此病危害程度随着马铃薯生产年份的不同而有差异。近年来 ,该病害在黑龙江省北部马铃薯生产区有上升趋势 ,严重的可造成2 0 %～ 4 0 %的减产。1　黑胫病的发生1 1　症状马铃薯黑胫病可发生在植株生长发育的任何阶段 ,表现出多种症状 ,主要危害地上茎、匍匐茎和块茎。被侵染的植株 ,茎呈现一种典型的墨黑色腐烂 ,病斑发展往往是从块茎开始经由匍匐茎传至茎基部 ,继而可发展到地上茎或扩展至全株。茎部发病往往使植株生长不良 ,…     

应用PCR技术快速检测马铃薯环腐病菌

根据马铃薯环腐病菌16SrDNA基因片段核苷酸序列设计引物(引物1∶5-′TGTACTCGGCCAT-GACGTTGG-3′和引物2∶5-′TACTGGGTCATGTTGGT-3)′,进行马铃薯环腐病菌PCR特异性扩增试验。合成的引物能从马铃薯环腐病菌总基因组DNA和细菌纯培养,以及人工接种和自然发病的马铃薯块茎中特异性扩增环腐病菌16SrDNA基因片段1046bp。该试验结果为马铃薯环腐病的鉴定、检测及病害流行学研究提供了新的技术和方法。     

马铃薯环腐病菌Real-time Taqman-PCR检测体系的建立

本研究根据环腐病菌16S rRNA保守序列设计特异性引物及探针,建立了马铃薯环腐病菌实时定量荧光PCR检测体系。研究表明,该体系可以准确、稳定、定量的检测出样品中最低浓度为1fg·μL-1(即3.21×103copies·μL-)1的目的基因,检测极限可以达到几个拷贝。该检测体系可对马铃薯环腐病菌进行微量检测,对于保证马铃薯各级种薯、商品薯及其相关产品的质量,提高市场竞争力具有重要意义。     

油菜黑胫病研究进展

油菜黑胫病是一种世界性分布的真菌病害,由子囊菌Leptosphaeria biglobosa引起。近年来随着田间调查的深入,及其姊妹病害油菜茎基溃疡病（Leptosphaeria maculans引起）传入风险的增加,我国对油菜黑胫病的重视程度逐年增加。本文对油菜黑胫病病原菌种群、病害循环及危害症状、分布与寄主范围、分子检测技术以及综合防治等方面的研究进展进行了综述,分析了油菜茎基溃疡病菌对我国的侵入风险,同时对我国油菜黑胫病研究和防控提出了建议,并就此进行了展望。     

估算马铃薯块茎细菌密度的方法

Kloepper(1983)曾报导,在栽植前对马铃薯种薯用荧光假单胞菌处理,减少了受黑胫病菌(Erwinia Carotovora)侵染子薯的比例。在本文这方面的研究中,需要一种能够确定每单位块茎表面积细菌数目的方法。在检测马铃薯根部细菌的集群现象时,     

马铃薯晚疫病种薯带菌的分子检测

从马铃薯块茎上分离纯化得到晚疫病菌、银腐病菌、癌肿病菌、红腐病菌、黑点病菌和镰刀菌等并提取它们的全基因组DNA,用引物08-3和08-4对这些真菌的DNA进行PCR扩增,以检验该引物扩增晚疫病菌DNA的特异性。取马铃薯种薯的芽眼、芽及其他不同部位并用NaOH法提取晚疫病菌DNA,再用引物08-3和08-4进行特异性扩增以检验种薯是否带晚疫病菌。结果表明:(1)引物08-3和08-4有很强的特异性,只能扩增出大小为257bp的马铃薯晚疫病菌DNA片段;(2)用该引物能扩增出种薯芽眼组织中大小为257bp的晚疫病菌DNA,说明通过PCR特异扩增,能直接检测出马铃薯种薯中潜伏的晚疫病菌。(3)本次检测的本地感病品种米拉有10%的种薯带晚疫病菌。该研究为种薯传播晚疫病菌的分子检测提供了依据和方法。     

山东省马铃薯病害调查

山东省是中国马铃薯种植大省,但是由于多年连作,导致马铃薯病害愈加严重。为探究山东省马铃薯常见病害发生情况,于2015年秋季采集了滕州、泰安和高密3个市区的44个样本,提取病株的DNA和RNA,分别对这些样本进行13种马铃薯常见细菌性病害、真菌性病害和病毒病的检测。结果表明,山东省发现的马铃薯病害主要有疮痂病、黑胫病、干腐病、黑痣病、病毒病PVX和PVY。这将为山东省马铃薯病害的防治提供一定的依据,同时也为马铃薯育种提供一定的参考价值。     

马铃薯病组织传播黑胫病的研究

田间试验表明 ,室外存放 3年的马铃薯黑胫病病组织播种时撒入播土内 ,具有较强的侵染性。马铃薯病根组织处理的发病率最高 (9 0 3% ) ,病茎叶和无症状植株两个处理均为 5 16 % ,比未撒入马铃薯病组织的对照增大 3～ 6倍 ,显然病组织对土壤传播马铃薯黑胫病具有重要作用     

The influence of inoculum density and environment on the development of potato blackleg

Significant increases in pre-emergence seedpiece decay and preemergence shoot infection were observed asErwinia carotovora var.atroseptica inoculum density increased. As inoculum density increased, a point was reached where virtually 100 per cent of the disease was expressed as preemergence decay and no typical postemergence blackleg occurred. Soil temperature and soil moisture markedly affected blackleg incidence, especially at low inoculum densities. Increasing soil temperature (from O°C to 30°C) and soil moisture (from 65 to 100% of field capacity) usually resulted in an increased total blackleg incidence. The effect of soil temperature and soil moisture appeared to be masked at high inoculum densities. Conditions such as low inoculum density and/or cool soil temperatures and low soil moistures which apparently slowed the rate of seedpiece decay usually increased the amount of postemergence blackleg, but also reduced the total amount of disease which occurred. It is suggested that conditions which are not optimal for the pathogen result in increased amounts of typical easily diagnosed postemergence blackleg expression, whereas conditions which favor the pathogen may reduce this phase of the disease but actually increase the total loss by increasing the amount of seedpiece decay and preemergence death of potato shoots.     

Expression of the chicken lysozyme gene in potato enhances resistance to infection byErwinia carotovora subsp.atroseptica

Infection of potato plants and tubers with the bacteriumErwinia carotovora subsp.atroseptica produces blackleg and soft rot diseases, which cause significant losses to crops and stored potatoes. In order to obtain resistance against this bacterium, the genechly encoding the enzyme lysozyme from chicken was introduced into potato plants (cv. Desirée) viaAgrobacterium- mediated transformation. Sixty-three and 69 transgenic potato clones were evaluated in the greenhouse for resistance to blackleg and soft rot diseases, respectively. Results reported in this paper indicate that 21%-29% of the potato clones showed increased resistance to infection by the bacteriumE. c. subsp.atroseptica T7, as revealed by a reduced severity of blackleg or soft rot symptoms. Nine clones showing different levels of resistance were selected for further molecular analysis. The number of copies of the transgene integrated in the plant genome of these clones was estimated by Southern blot analysis. The level of transgene expression, detected by Northern blot analysis, correlated with the level of resistance detected in these clones.     

热分析对马铃薯抑芽粉剂中氯苯胺灵缓释机理的研究

采用热重法、导数热重法和差热分析法,分别对基料凹凸棒、主效成分氯苯胺灵和抑芽粉剂进行热分析测定,通过对热分析结果的比较,提出了基料凹凸棒对主效成分氯苯胺灵的缓释机理。结果表明,经过液化工艺将氯苯胺灵附载到凹凸棒上,由于基料凹凸棒的比表面积较大,吸附在凹凸棒上的氯苯胺灵因表面效应在较低的温度下也易于挥发,并延缓了挥发速率,为选用凹凸棒作为基料对氯苯胺灵的缓释提供了理论依据。     

马铃薯病毒外壳蛋白融合基因转化马铃薯及其抗病性分析

将多种病毒的有效核酸片断拼接成融合基因转入马铃薯可获得多抗马铃薯材料。针对马铃薯生产中分布广泛、危害严重并经常混合感染的马铃薯X病毒(PVX)、马铃薯Y病毒(PVY)、马铃薯卷叶病毒(PLRV)和马铃薯S病毒(PVS),开展了利用基因工程方法获得兼抗4种马铃薯病毒转基因马铃薯材料的研究。试验在前期获得含4种马铃薯病毒外壳蛋白基因片段的质粒pART27-XSYV-rh的基础上,通过根癌农杆菌(Agrobacterium tumefaciens)介导转化马铃薯(Solanum tuberosum)品种‘陇薯3号’,PCR扩增和PCR-Southern杂交证明,4价融合基因已整合到马铃薯基因组中。qRT-PCR分析表明,该融合基因在转基因植株中能正常表达。3株转基因植株的抗病性鉴定结果表明,2株对4种病毒同时具有抗性;1株对PLRV侵染表现阳性,对另外3种病毒同时具有抗性。     

Identification of late blight, Colorado potato beetle, and blackleg resistance in three Mexican and two South American wild 2x (1EBN)Solanum species

Wild potatoes are important sources of genes for resistance to disease and insect pests. A collection of wild Mexican and South AmericanSolarium species from the US potato Genebank was evaluated under laboratory and/or field conditions for their reaction to late blight (Phytophthora infestans), Colorado potato beetle (CPB,Leptinotarsa decemlineata Say), and blackleg (Erwinia carotovora subsp.atroseptica (van Hall) Dye) in order to identify individual genotypes with multiple resistance genes. Late blight inoculations using aggressive isolates (US-8/A2 and US-11/A1 mating types) of P.infestans revealed a wide range of variation for resistance between and within the accessions of the wild species tested. For late blight, susceptible as well as moderately to highly resistant genotypes were observed in all the species tested. However, at least one accession from the three Mexican and one South American wild diploid species tested showed a relatively uniform high level of resistance toP. infestans. These includedS. bulbocastanum, S. pinnatisectum, S. cardiophyllum, andS. circaeifolium. Two accessions from South American speciesS. commersonii were highly susceptible to late blight. For the Colorado potato beetle test, only one species,S. pinnatisectum appeared uniformly resistant to CPB under field conditions. Results of screening for blackleg resistance showed that there were major differences between genotypes in the wild species. Accessions ofS. circaeifolium PI 498119 andS. bulbocastanum PI 243504 were identified as having significantly higher blackleg resistance than cultivated potato and the other wild species tested. However, genotypes from these two accessions were more susceptible to late blight and CPB. Characterization of theP. infestans isolate P1801C.16 used for late blight evaluation and multi-locus isolate tests using US-8/A2 and US-11/A1 races revealed that the resistance inS. pinnatisectum genotypes tested corresponded to a race-non-specific genetic system, which was different from any existing R genes.Solanum pin-natisectum genotypes with both high levels of late blight and CPB resistance as well as blackleg resistance genotypes identified in the present study represent a diverse gene pool that may be useful for development of new potato cultivars with multiple disease and insect resistance. The potential utilization of these valuable sources for improvement of cultivated potato is discussed.     

马铃薯新品种（系）在大理州早春作的适应性评价

通过对引进的马铃薯新品种（系）进行适应性评价,筛选出适合大理早春作大面积推广种植的马铃薯新品种,为该区马铃薯品种更新换代提供科学依据。以‘合作88’为对照,对比了5个新品种（系）的植株形态特征,抗性表现,鲜薯产量及块茎品质。结果表明：‘同薯20-2’出苗早,出苗率高,茎叶浓绿,植株茂盛,感病相对较轻且产量、商品薯率、干物质含量、淀粉含量均高于其他参试品种。经进一步试验后可在该地区推广种植。     

Enhanced resistance to bacterial infection byErwinia carotovora subsp.atroseptica in transgenic potato plants expressing the attacin or the cecropin SB-37 genes

Blackleg and soft rot diseases, caused by the bacteriumErwinia carotovora, are among the diseases that cause important losses in culture and storage of potato. In this paper, we introduced bacterial resistance into potato, via genes encoding for proteins with antibacterial activity. For this purpose, potato clones were transformed either with the gene encoding the acidic attacin protein fromHyalophora cecropia, or with the gene encoding the cecropin analog peptide SB37. These clones were evaluated for soft rot and blackleg resistance, after inoculation with the bacterial strainErwinia carotovora subsp.atroseptica T7. Results reported in this paper indicate that a considerable percentage of the potato clones (15–22%) showed increased resistance to bacterial infection, revealed by reduced severity of blackleg or soft rot symptoms. Expression of the transgenes was demonstrated in some of the clones by Northern blot analysis. This is the first report indicating that expression of the gene encoding for an attacin protein and for the cecropin SB-37 peptide in transgenic potato confers increased resistance to bacterial infection.     

Long-day-adaptedSolanum phureja as a source of resistance to blackleg caused byErwinia carotovora subsp.atroseptica

Summary Long-day-adaptedSolanum phureja clones were assessed for resistance to blackleg caused byErwinia carotovora subsp.atroseptica under field and controlled environmental conditions over two years. In the field, twenty-two of the twenty-three clones ofS. phureja assessed were as resistant to blackleg as the commercial cultivar Ailsa, the most resistant control, and were significantly (P<0.001) more resistant than the intermediate and susceptible cultivars Wilja and Estima, respectively. Under controlled environmental conditions, resistance in commercial cultivars was more easily overcome. However, 18 of the 21S. phureja clones assessed were significantly more resistant to blackleg than these cultivars.     

马铃薯晚疫病预警技术发展历史与现状

本文系统介绍了国内外马铃薯晚疫病预警技术的发展历史和应用现状。马铃薯晚疫病预警技术的发展大体经历了三个阶段,第一阶段是基于一定气象条件规则的人工预警方法;第二阶段是基于预测模型的电算预警技术;第三阶段是基于田间病害监测和信息技术的监测预警系统。目前,一些发达国家特别是欧洲主要应用监测预警系统。我国也已组建了"中国马铃薯晚疫病监测预警系统",并已实现网上运行和服务(其域名为:www.china-blight.ne)t,该系统的进一步推广应用,将为我国马铃薯晚疫病监测预警和有效防控提供有力保障。