甘肃省马铃薯粉痂病品种抗病性及防治药剂筛选

由马铃薯粉痂病菌Spongospora subterranea引起的粉痂病已成为甘肃省马铃薯生产上的重要病害之一,发生面积及危害程度均呈逐年增加的趋势,严重威胁马铃薯产业的健康可持续发展。研究马铃薯不同品种对粉痂菌的抗性并筛选高效防治药剂,是控制该病扩散蔓延最为直接和有效的手段,对马铃薯粉痂病的综合治理具有重要意义。本试验对甘肃省马铃薯主栽品种对粉痂病抗病性及田间防治药剂开展了系统研究,抗病性测定结果表明：22个品种块茎上均可感染粉痂病,发病率为25.00%～100.00%,病情指数为6.67～68.33,平均病情指数为29.45。从发病率和病情指数综合评价,‘冀张薯12号’对粉痂病有较好的抗性。田间药剂筛选试验结果表明：10亿cfu/g多黏类芽孢杆菌可湿性粉剂100倍液和0.3%四霉素水剂50倍液播前种薯浸种处理均对马铃薯粉痂病有较好的防治效果,防效分别为60.50%和60.22%,显著高于其他处理;0.3%四霉素水剂30倍液和10亿cfu/g多黏类芽孢杆菌可湿性粉剂45倍液于马铃薯花期时进行灌根处理,对粉痂病的防效分别为56.00%和52.87%,显著高于其他处理。因此,甘肃省马铃...     

云南省会泽县马铃薯粉痂病发生情况调查分析初报

马铃薯粉痂病[Spongospora subterranea (Wallr.) Lagerh.]是近年在会泽县新发生的病害,在高温、多雨、品种感病的情况下发病严重,不但影响马铃薯的产量,更重要的是影响马铃薯的商品性和病原菌传播。为此,本县植保站根据本县马铃薯的种植区域和品种布局,开展了不同品种、不同土壤及不同海拔种植区马铃薯粉痂病发病程度调查,旨在为当地马铃薯粉痂病的研究和防治提供依据。     

双重PCR检测马铃薯晚疫病和环腐病方法的建立

通过克隆马铃薯环腐病菌和晚疫病菌转录间隔区(ITS)序列,并对测序结果进行同源性比较,选取差异位点分别设计了两对引物P.IN1/P.IN2和C.IN1/C.IN2,并检测了引物的特异性及方法的灵敏度。引物P.IN1/P.IN2可扩增出1条363bp马铃薯晚疫病菌的特异性条带,在DNA水平上其灵敏度达18fg/μL;引物C.IN1/C.IN2可扩增出1条218bp马铃薯环腐病菌的特异性条带,在细菌数上检测灵敏度为104 cfu/mL。混合这两对引物构建双重PCR反应体系,能从马铃薯环腐病菌和晚疫病菌的混合DNA及感染这两种菌的马铃薯植株中同时扩增到363bp和218bp的特异片段。实现了同时对马铃薯晚疫病菌和环腐病菌的快速可靠检测。     

云南马铃薯粉痂病病原研究

马铃薯粉痂菌(Spongospora subterranea f.sp.subterranea)是引起马铃薯粉痂病的病原。本试验对云南省马铃薯产区的马铃薯粉痂菌进行了研究,通过光学和扫描电子显微镜对病原菌进行了描述。光学显微镜观察到了典型的海绵状蜂窝结构;扫描电子显微镜观察到的休眠孢子囊结构与国外观察的一致,证明了马铃薯粉痂菌在云南省马铃薯主产区的存在。     

双重PCR检测马铃薯晚疫病菌和青枯病菌方法的建立及应用

 利用真菌通用引物ITS1和ITS4扩增马铃薯晚疫病菌转录间隔区并进行序列测定,通过序列比较,设计了1对马铃薯晚疫病菌的特异引物INF1/INF2,并对15种不同真菌、细菌和7种疫霉属和腐霉属卵菌基因组DNA进行PCR扩增,结果只有不同来源的马铃薯晚疫病菌株可获得324 bp的特异带。将引物INF1/INF2与卵菌通用引物进行巢式PCR扩增后,其检测灵敏度在DNA水平上可达30 fg。运用设计的引物与马铃薯青枯病菌特异引物结合建立了双重PCR体系,能从马铃薯晚疫病菌和马铃薯青枯病菌总基因组DNA以及人工接种和自然发病的马铃薯植株中分别或同时扩增到324 bp和281 bp的特异片段。实现了同时对马铃薯晚疫病菌和马铃薯青枯病菌的快速可靠检测。     

马铃薯干腐病优势病原菌Fusarium sambucinum 分子检测技术的建立

马铃薯干腐病是马铃薯最重要的贮藏期病害之一,现已成为马铃薯贮藏期烂窖的重要原因。实现病原菌的快速检测对病害诊断和科学防控具有重要的实践意义。本研究基于镰刀菌翻译延伸因子序列设计了一对检测接骨木镰刀菌Fusarium sambucinum的特异引物Fs-F/Fs-R。该特异引物可从接骨木镰刀菌中获得309bp的特异性扩增片段,而其他种类镰刀菌及马铃薯重要病害病原菌中均无此片段,说明该引物具有专一性。该体系的灵敏度检测结果表明,最低能检测到的接骨木镰刀菌基因组DNA浓度为70pg/μL。该引物也适用于发病马铃薯块茎中接骨木镰刀菌的快速检测。     

云南省马铃薯粉痂病的发生与危害调查分析

粉痂病严重影响马铃薯的产量和品质,是我国马铃薯产业健康发展的重要制约因素之一.为进一步探明马铃薯粉痂病在云南省的发生、分布及危害情况,本研究于2018年-2019年,在云南省内马铃薯不同生态种植区,开展了马铃薯粉痂病的发生与危害情况调查.调查范围涉及19个县(区)、涵盖了19个当地马铃薯主栽品种(系).调查结果显示:粉...     

马铃薯粉痂病综合防治技术初探

近年来,粉痂病已成为云南省马铃薯生产上的重要病害之一,但目前还没有有效防治粉痂病的药剂和措施。本试验于2005年对云南省马铃薯粉痂病的综合防治技术进行了大田试验和温室盆栽试验。结果表明:在大田试验和温室盆栽试验中,品种会-2对马铃薯粉痂病均表现较高的抗性,在播种穴中施用适量的豆饼进行处理均能较好地防止粉痂病的发生,其他处理的防治效果则不明显。     

马铃薯青枯病的PCR检测

以我国11个省(市、区)和6种不同寄主植物的43个青枯菌Ralstonia solanacearum代表性菌株和4个国外青枯菌菌株为试材,采用15条随机引物进行了RAPD分析,筛选到了1条青枯菌所特有的DNA片段,根据其碱基序列,设计了特异PCR引物,对不同青枯菌DNA进行扩增,并以马铃薯的其它病原细菌为对照,只有青枯菌可获得773bp的DNA扩增产物.经过对PCR反应模板制备程序的简化和优化,利用本研究设计的特异引物,直接对马铃薯病块茎的DNA粗提液进行扩增,获得了773bp片段.此技术可望用于马铃薯种薯青枯病菌的检测,大大缩短检测时间,提高检测效率.     

双重RT-PCR同步检测马铃薯A病毒和马铃薯卷叶病毒

根据马铃薯A病毒全基因序列和马铃薯卷叶病毒衣壳蛋白区序列分别设计PVA和PLRV的特异性引物,应用双重RT-PCR同步检测马铃薯A病毒和马铃薯卷叶病毒,分别得到300bp和222bp大小的扩增片段.试验从反转录、PCR等方面对双重RT-PCR同步检测2种病毒进行了探索和优化.结果表明反转录反应中dNTPs浓度、2种病毒下游引物浓度比例以及PCR反应中Mg2+浓度对双重RT-PCR同时检测PVA和PLRV有较大的影响.     

云南省马铃薯疮痂病致病链霉菌种类组成研究

 为明确云南省马铃薯疮痂病病原菌(Streptomyces spp.)的种类及其生物学特征,自2013年从云南省13个马铃薯主产区采集疮痂病病样,共分离到200株链霉菌,通过温室盆栽致病性试验筛选出67株致病菌。通过形态学、生理生化指标、致病性测定及16S rDNA序列分析对获得的菌株进行鉴定。结果显示,引起云南地区马铃薯疮痂病的病原为10种链霉菌,分别为S. caviscabies、S. anulatus、S. scabies、S. turgidiscabies、S. acidiscabies、S. europaeiscabiei、S. luridiscabiei、S. enissocaesilis、S. griseus和S. aureofaciens。其中S. enissocaesilis和 S. anulatus为优势种群,S. caviscabies、S. anulatus和S. luridiscabiei为国内首次报道的病原菌。因此,认为云南省马铃薯疮痂病菌种类复杂多样。     

Effect of soil inoculum level and environmental factors on potato powdery scab caused by Spongospora subterranea

The effects of soil inoculum level and three environmental factors (soil type, soil moisture regime and temperature) on the incidence and severity of powdery scab caused by Spongospora subterranea were investigated in potato plants grown under controlled environmental conditions. Symptoms of powdery scab on tubers were assessed visually, after which DNA was extracted from tuber peelings and quantified in a real-time polymerase chain reaction assay using primers and a TaqMan® probe specific to S. subterranea to establish tuber infection levels. Soil inoculum concentration of S. subterranea did not significantly affect the incidence and severity of either tuber infection or powdery scab symptoms at maturity. No significant differences in disease incidence and severity were found between sandy, loamy and clay soils, although the two lighter soils yielded more powdery scab than clay soil. Constant dampness of the soil resulted in significantly more disease than a fluctuating moisture regime. Infection and disease levels were high at all three temperatures tested (9, 12 and 17°C), but symptoms were most severe at 12°C. The percentage of plants with infected tubers did not increase after tuber initiation, although the amount of S. subterranea DNA detected in tubers and the severity of powdery scab symptoms increased in mature plants. Latent tuber infections were found to be common, especially under conditions suboptimal for disease development. This new information may be important for the prevention of powdery scab in potato-growing areas around the world.     

Occurrence and survival of potato scab pathogens (Streptomyces species) on tuber lesions: quick diagnosis based on a PCR-based assay

A time-saving and cost-effective polymerase chain reaction (PCR)-based method was developed for species-specific detection of the scab pathogens ( Streptomyces scabies and S. turgidiscabies ) prevalent in potato ( Solanum tuberosum ) in northern Scandinavia. Species specificity of primers was verified using a collection of previously characterized Streptomyces strains isolated from potato scab lesions in Finland and Sweden. A total of 1245 scab lesions was tested from potato cvs Matilda and Sabina grown in the field in two geographic regions of Finland in 2000 and 2001. Freshly harvested or stored potato tubers were incubated at room temperature (18–21°C) under humid conditions for a few days. Bacterial growth was collected from scab lesions for DNA isolation and PCR. The two scab pathogens were detected in the same potato fields, tubers and scab lesions. The relative incidence of S. scabies was high in freshly harvested tubers but was much lower than that of S. turgidiscabies following storage. Both pathogens were seed-transmitted in Matilda and Sabina after 24 weeks of storage at 4°C.     

Biocontrol of powdery scab of potato by seed tuber application of an antagonistic fungus, <Emphasis Type="Italic">Aspergillus versicolor</Emphasis>, isolated from potato roots

An environmentally friendly measure to control potato powdery scab caused by a protozoan pathogen Spongospora subterranea f.sp. subterranea (Sss) was developed by focusing on antagonistic microorganisms that were considered compatible with potato root. Five hundred and eight soil fungi, isolated from potato root cultivated in soil suspensions from four potato fields in Hokkaido, were screened for suppressiveness of root infection by Sss in a hydroponic culture system and for powdery scab severity in greenhouse and field experiments. Antagonistic isolate Im6-50, identified as Aspergillus versicolor, was selected as a potent biological control agent. In a 3-year field test, A. versicolor Im6-50 suppressed powdery scab with a protection value of 54–70 (100?=?complete protection) when applied directly on seed tubers compared with a protection value of 77–93 by the synthetic fungicide fluazinam. A. versicolor Im6-50 was detected from the surface of daughter tubers and from the soil in which the inoculated seed tubers were cultivated by PCR using species-specific primers. The establishment of A. versicolor Im6-50 on the stolon of inoculated potato plants and in the rhizosphere is considered to contribute to the mechanism for disease suppression.     

茄匍柄霉菌实时荧光定量PCR检测方法的建立及应用

为了准确检测病残体内茄匍柄霉菌(Stemphylium solani)DNA含量在土壤内的动态变化,本研究根据三磷酸甘油醛脱氢酶(GAPDH)基因序列,设计并筛选特异性引物Stem-g7F/Stem-g7R,能从靶标基因组DNA中特异性扩增出大小为150 bp的目的片段.建立的Stemphylium solani实时荧...     

Detection of Colletotrichum coccodes from soil and potato tubers by conventional and quantitative real-time PCR

Colletotrichum coccodes is the causal agent of the potato blemish disease black dot. Two PCR primer sets were designed to sequences of the ribosomal internal transcribed spacer (ITS1 and ITS2) regions for use in a nested PCR. The genus-specific outer primers (Cc1F1/Cc2R1) were designed to regions common to Colletotrichum spp., and the species-specific nested primers (Cc1NF1/Cc2NR1) were designed to sequences unique to C . coccodes . The primer sets amplified single products of 447 bp (Cc1F1/Cc2R1) and 349 bp (Cc1NF1/Cc2NR1) with DNA extracted from 33 European and North American isolates of C. coccodes. The specificity of primers Cc1NF1/Cc2NR1 was confirmed by the absence of amplified product with DNA of other species representing the six phylogenetic groups of the genus Colletotrichum and 46 other eukaryotic and prokaryotic plant pathogenic species. A rapid procedure for the direct extraction of DNA from soil and potato tubers was used to verify the PCR assay for detecting C. coccodes in environmental samples. The limit of sensitivity of PCR for the specific detection of C. coccodes when inoculum was added to soils was 3·0 spores per g, or the equivalent of 0·06 microsclerotia per g soil, the lowest level of inoculum tested. Colletotrichum coccodes was also detected by PCR in naturally infested soil and from both potato peel and peel extract from infected and apparently healthy tubers. Specific primers and a TaqMan fluorogenic probe were designed to perform quantitative real-time (TaqMan) PCR to obtain the same levels of sensitivity for detection of C. coccodes in soil and tubers during a first-round PCR as with conventional nested PCR and gel electrophoresis. This rapid and quantitative PCR diagnostic assay allows an accurate estimation of tuber and soil contamination by C. coccodes .     

小麦纹枯病菌Rc-RPA-LFD快速检测方法的建立及应用

Rhizoctonia cerealis is a soil-borne phytopathogenic fungus that causes wheat sharp eyespot, resulting in serious economic losses. In this study, the recombinase polymerase amplification combined with lateral-flow dipstick technology (Rc-RPA-LFD) was developed for the rapid and sensitivity detection of R. cerealis. The Rc-RPA-LFD assay could be completed at isothermal temperature of 38°C within 30 min without PCR thermal cyclers. The RPA primers and probe designed based on ITS region, showed high specificity to R. cerealis. The detection limit of Rc-RPA-LFD assay was 1 pg·μL^-1 fungal genomic DNA, showed an equal sensitivity to that of conventional PCR. In addition, the Rc-RPA-LFD assay could detect R. cerealis from field soil samples, showing no significant differences compared to conventional PCR assay. The simplicity, rapidly and practicability all indicated that Rc-RPA-LFD assay will be a promising molecular diagnosis for the accurate and rapid detection of R. cerealis.     

小麦纹枯病菌Rc-RPA-LFD快速检测方法的建立及应用

正近年来,随着气候条件、耕作制度的变化和水肥条件的改善,小麦纹枯病在我国危害日趋严重,现已成为长江中下游麦区和黄淮麦区主要病害,每年造成严重经济损失[1]。小麦纹枯病常与小麦根茎部病害如根腐病、茎基腐病混合发生,发病早期症状相似,影响病害的早期诊断和防治。目前,小麦纹枯病菌快速检测方法主要基于常规PCR和Real-time PCR [3,4]。常规PCR和Real-time PCR具备检测准确、灵敏度高的优点,但这两种检测技术对仪器设备和     

Using the TxtAB operon to quantify pathogenic Streptomyces in potato tubers and soil

The phytotoxin thaxtomin, produced by plant pathogenic Streptomyces species, is the only known pathogenicity determinant for common scab diseases of potato and other root and tuber crops. Genes encoding thaxtomin synthetase (txtAB) are found on a pathogenicity island characteristic of genetically diverse plant pathogenic Streptomyces species. In this study, an SYBR Green quantitative real-time polymerase chain reaction (PCR) assay using primers designed to anneal to the txtAB operon of Streptomyces was developed to quantify pathogenic bacterial populations in potatoes and soil. The real-time PCR assay was specific for pathogenic Streptomyces strains. The detection limit of the assay was 10 fg of the target DNA, or one genome equivalent. Cycle threshold (Ct) values were linearly correlated with the concentration of the target DNA (correlation coefficient R(2) = 0.99) and were not affected by the presence of plant DNA extracts, indicating the usefulness of the assay for quantitative analyses of the pathogenic bacteria in plant tissues. The amount of pathogenic Streptomyces DNA in total DNA extracts from 1 g asymptomatic and symptomatic tubers was quantified using the assay and ranged from 10(1) to 10(6) pg. A standard curve was established to quantify pathogenic Streptomyces in soil. Using the standard curve, numbers of pathogenic Streptomyces colony forming units were extrapolated to range from 10(3) to 10(6) per gram of soil from potato fields where common scab was found. This real-time PCR assay using primers designed from the txtAB operon allows rapid, accurate, and cost effective quantification of pathogenic Streptomyces strains in potato tubers and in soil.     

马铃薯疮痂病菌在植株和田间的分布与动态分析

为探明马铃薯疮痂病菌在植株和土壤中的分布情况及种群动态变化特点,利用常规PCR和定量PCR(qPCR)技术对不同环境的马铃薯疮痂病株和田间植株不同生育期的土壤样品进行病原菌的定性定量检测.结果 表明,病田、温室盆栽和微型薯苗床中马铃薯疮痂病重度发病植株的根、匍匐茎、块茎、地上茎、叶片等组织样品均可检测到184 bp的疮...