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。     

嵌套式PCR超灵敏检测马铃薯环腐病菌

马铃薯种薯中存在环腐病菌潜伏侵染,这种潜伏侵染逐代累积、逐渐表现症状,这是马铃薯环腐病无法根除的根本原因。本研究应用国外成功报道的根据存在于pCS1质粒和环腐菌染色体中一个1.3 kb的插入因子IS1121、高度重复的DNA片段设计的环腐病菌亚种特异性引物序列合成出引物对CMSIF1-CMSIR1和引物对CMSIF2-CMSIR2。以环腐标准菌株、黑龙江省环腐菌株以及马铃薯上其它主要的细菌病原菌(青枯病、软腐病、黑胫病)为试验材料进行直接PCR和嵌套式PCR扩增,结果只有环腐标准菌株和黑龙江省环腐菌株出现特异性片段(直接PCR扩增出1046 bp的片段,嵌套式PCR扩增出864 bp的片段)。将环腐菌纯菌种菌悬液稀释成浓度梯度并与马铃薯组织液混合进行直接PCR和嵌套式PCR检测灵敏度比较,结果表明嵌套式PCR检测灵敏度比直接PCR检测灵敏度提高了100￣1000倍。以明显感病症状的块茎、无明显感病症状的块茎和健康块茎为试验材料进行直接PCR和嵌套式PCR,结果除明显感病症状块茎外,所有无明显感病症状的块茎也均被检测出带有环腐病菌。     

马铃薯接骨木镰刀菌的分子检测

接骨木镰刀菌是马铃薯干腐病的主要致病菌,给黑龙江省马铃薯造成了一定的经济损失。采用马铃薯干腐病引物LDJ1对马铃薯干腐病、马铃薯晚疫病、马铃薯早疫病、马铃薯黑痣病的致病菌的ITS序列进行测定,只有马铃薯干腐病菌扩增出560 bp特异性片段。再分别对接骨木镰刀菌、燕麦镰刀菌和茄病镰孢变种的ITS序列进行BLAST比对分析,针对接骨木镰刀菌设计了特异引物L2,扩增特异性产物为278 bp,该引物所建立的PCR检测体系可检测DNA含量在50 pg/μL以上的目的基因。     

马铃薯卷叶病毒CP基因的RT-PCR扩增

根据马铃薯卷叶病毒CP基因的序列设计合成了两对特异性DNA引物,用RNA提取试剂RNAplant从感病的马铃薯叶片中提取总RNA,在3′引物引导下以总RNA为模板反转录合成cDNA第一链,然后进行PCR,分别扩增出了长627 bp和336 bp的特异性PCR产物,其大小与预期的CP基因及其部分序列一致,实现了马铃薯卷叶病毒CP基因及其片段的简便、有效的RT-PCR扩增。     

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。     

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。     

甘蔗黑穗病菌和赤腐病菌双重PCR检测体系的建立

目前,针对甘蔗黑穗病菌与赤腐病菌单个病菌检测体系已相继建立,但是仍然未见两个病原菌的双重PCR检测方法。在本研究中,根据黑穗病菌交配型b E基因、甘蔗赤腐病菌特异性SCAR片段序列的特异性引物,在二者单一PCR检测体系的基础上,建立并优化可同时检测黑穗病菌和赤腐病菌的双重PCR检测方法。建立的双重PCR检测体系可从黑穗病菌与赤腐病菌样品中分别扩增出320 bp和442 bp的特异条带。灵敏度分析结果表明,对混合模板黑穗病菌与赤腐病菌的最低检测水平量均为0.1ng。     

烟草青枯病菌巢式PCR检测方法的建立及应用

利用细菌16S-23S r DNA内源转录间隔区通用引物L1/L2扩增烟草青枯病菌基因组DNA,并对其扩增产物进行克隆测序,经与近缘种序列多重比对分析后,设计1对特异性引物Rs F/Rs R,用于包括烟草青枯病菌在内的15种不同细菌、5种真菌、3种卵菌基因组DNA的PCR扩增。结果表明:在优化的反应体系与程序条件下,该对引物只能从烟草青枯病菌中扩增出241 bp的特异片段,并通过序列测定验证了其准确性;将引物Rs F/Rs R与细菌通用引物L1/L2进行巢式PCR扩增后,其检测灵敏度在DNA水平上可达0.4 fg/μL,较常规PCR提高1 000倍,表明该对引物能有效地用于烟草组织及土壤中青枯病菌的检测。此结果对烟草青枯病的早期诊断、快速检测及病害流行学研究具有重要意义。     

实时定量荧光PCR法检测马铃薯黑胫病菌

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,严重地影响了马铃薯的产量和质量。本研究根据Potato Black Leg序列,设计出马铃薯黑胫病菌特异性的引物,对10种供试菌株进行了实时荧光PCR检测。结果表明,该方法的特异性好,可以将马铃薯黑胫病菌和其它马铃薯常见病害相区分;灵敏度较高,可检测出最低的黑胫病菌浓度为3.6~3.9 cfu.mL-1;而且检测时间仅用4 h,大大缩短了检测周期。该方法可有效地应用于马铃薯黑胫病菌的检测和监控。     

不同品种（系）马铃薯休眠块茎顶端和茎端芽眼组织马铃薯Y病毒含量分析

马铃薯Y病毒（Potato virus Y,PVY）是马铃薯生产中较常见的病毒之一,马铃薯病毒的检测是生产中保障马铃薯品质和产量的重要基础,收获后批量检测是进行种薯质量评价的主要依据之一。研究针对‘56-2’‘Ivory Russet’‘Clearwater Russet‘’龙薯14号‘’龙薯3号‘’龙薯15号‘’龙育201401-70‘’龙薯7号‘’克新23号’和‘克新28号’马铃薯品种（系）休眠块茎样品,采用TRIzol法提取马铃薯休眠块茎顶端和茎端芽眼组织的总RNA,经qRT-PCR和RT-PCR检测分析,比较块茎的顶端与茎端芽眼组织PVY含量分布的情况。结果表明,上述供试材料的休眠块茎茎端芽眼组织PVY浓度均高于顶端芽眼组织,能够精准的检测到病毒的存在;对‘Ivory Russet’品种的休眠块茎选取100个带病样品通过TRIzol法提取顶端与茎端的总RNA,采用4合1的方法合样后进行PVY RT-PCR检测,所有处理的顶端芽眼组织检测条带亮度低于茎端芽眼组织。可见,马铃薯休眠块茎茎端芽眼组织取样能够更加精准的检测出PVY。研究结果为马铃薯种薯收获后PVY的检测及合理取样提供了...     

Discrimination of three fungal diseases of potato tubers based on volatile metabolic profiles developed using GC/MS

Summary Volatiles from the headspace of Russet Burbank potato tubers, non-wounded non-inoculated (N-control), wounded-inoculated-with sterile water (W-control), wounded-inoculated withPhytophthora infestans, Pythium ultimum orBotrytis cinerea, respectively, were sampled at 3 and 6 days after inoculation (dai), using gas chromatography/mass spectrometry (GC/MS).Botrytis inoculated tubers produced two specific volatiles: 2-2-propenyl-l,3-dioxolane and 3, 5-heptadiyn-2-one, while thePythium inoculated tubers produced three: 2-methyl-l-butanol, 2-butanone and 2-methyl-2-butanamine. Similarly, ethoxy-ethene was specific forPhytophthora inoculated tubers. 5-l-methylethylidene-l,3-cyclopentadiene was specific to W-control tubers. Discriminant analysis models based on metabolic fingerprints of metabolites or of mass ions correctly classified 80 to 100% of the observations into respective inoculations/diseases. However, a test-validation correctly classified only 44, 50, 44, 50 and 44% of the fingerprints based on consistent metabolites and 44, 63, 38, 75 and 31% of fingerprints based on mass ions, intoBotrytis, N-control,Phytophthora, Pythium and W-control, respectively. The disease discriminatory metabolite markers and the discriminant models developed here can be used to differentiate the three diseases of Russet Burbank potato tubers, after further validation under commercial conditions.     

Ion chromatography analysis of phosphite uptake and translocation by potato plants: Dose-dependent uptake and inhibition of Phytophthora infestans development

Phosphite-based fungicides are increasingly used to control fungi-like plant pathogens from the Oomycetes group. A rapid, precise, and cost-effective suppressed conductivity high performance ion chromatography (HPIC) method was developed to assess the concentrations of soluble phosphites (Phi) and phosphates (Pi) in plant samples. This technique was used to determine the amount of Phi and Pi in leaves and tubers of potato plants following foliar applications of the Phi-based fungicide Confine™. High amounts of Phi were determined in both leaves and tubers indicating that potato plants efficiently uptake and translocate the fungicide. The number of applications of Confine™ and its concentration were found to be directly proportional with the amount of Phi detected in potato plants and inversely proportional with the development of Phytophthora infestans (Mont.) de Bary in these plants. Levels of Phi comparable to those determined in plants were found to strongly inhibit the growth of P. infestans in vitro. The simultaneous estimation of the in planta Phi concentration and of the sensitivity of P. infestans to Phi represent the most comprehensive approach of assessing the efficacy of Phi-based fungicides in controlling late blight development in potatoes.     

Application of a PCR-based test for detection of potato late blight and pink rot in tubers

A polymerase chain reaction (PCR)-based test for potato late blight (Phytophthora infestans) and pink rot (P.erythroseptica, P. nicotianae) diseases has been developed for use with potato tuber tissue. Primers based on sequence analysis of the ITS2 region of ribosomal DNA of late blight and pink rot pathogens were utilized in PCR assays of inoculated tubers and tubers harvested from plots known to have late blight and/or pink rot. Assays of artificially inoculated Kennebec and Russet Burbank tubers revealed thatP. infestans was detected by PCR as early as 72 h after inoculation and in the absence of visible symptoms. Much higher detection frequencies were obtained by PCR compared with plating on selective medium or placement of tissue in moist chambers. Tubers from plots known to have late blight and/or pink rot were tested using the PCR assay. Assay of late blight lesions showed ca. 80% recovery for late blight-infected tubers from the field. Results indicate that the PCR assay provides a rapid and accurate test for diagnosis of late blight and pink rot in potato tubers.     

β-氨基丁酸诱导马铃薯叶片晚疫病抗性适宜浓度筛选

β-氨基丁酸(DL-β-aminobutyric acid,BABA)是一种非蛋白氨基酸,能有效诱导多种植物产生对多种病原菌的抗性。为了解BABA处理浓度和时间对马铃薯叶片晚疫病抗性的诱抗效果,本试验用四个浓度梯度(0.5mmol·L~(-1)、1mmol·L~(-1)、2mmol·L~(-1)和4mmol·L~(-1)的BABA喷雾处理马铃薯植株,处理1d、2d、3d和4d后分别取离体叶片接种晚疫病菌。结果表明:在0.5～4mmol·L~(-1)范围,随着BABA浓度增加和处理时间的延长,叶片抗性逐渐增强。2mmol·L~(-1)和4mmol·L~(-1)BABA处理3d后接种,离体叶片晚疫病抗性显著提高。     

Dormancy and sprout development of some South African potato cultivars during cold storage

Summary Observations were made on dormancy and sprout growth of nine potato cultivars stored at 3–4°C, 7–8°C and 11–12°C, respectively. Tubers of the cultivar Vanderplank had a very long dormant period (232 days at 3–4°C) and showed little sprout growth at 180 days. The cultivar Koos Smit had a very short dormant period (92 days at 3–4°C) and developed considerable sprout growth at the higher temperatures. The reaction of tubers of Up-to-date and BP₁ were approximately the same, and intermediate between those of Vanderplank and Koos Smit.     

Using Ethylene Gas and Chlorpropham Potato Sprout Inhibitors Together

Marketplace preference for lower pesticide residues in foods has led to research to reduce the residue of chlorpropham (isopropyl N-3-chlorophenyl carbamate; CIPC), a postharvest-applied sprout inhibitor which is widely used around the globe to prevent sprouting of stored potatoes (Solanum tuberosum L.). Ethylene gas, an effective, safe and non-toxic sprout inhibitor used in several countries, sometimes has negative effects on the colour of processed potato products when used alone. Trials were conducted over 3 years using cv. Shepody (French fry) and cv. NorValley (potato chips/crisps) to determine whether a combination of these two sprout inhibitors, at reduced dosages, could inhibit sprouting while maintaining good processing colour. CIPC applied at 0, 0.1, 0.25 and 1.0 times the recommended dosage was combined with 4 μll⁻¹ of ethylene gas applied or no ethylene at all (0 ethylene), for 1 day in 4 days, for 1 day in 2 days or continuously, in a factorial design. Sprout inhibition in both cultivars was excellent at all levels of CIPC application except the 0 rate. In both cultivars, sprouting was inhibited by the continuous ethylene treatment. However, all levels of ethylene exposure except the 0 rate negatively affected processing colour in both cultivars. The darkening was dose dependent, whereby the colour was darkest in continuous ethylene and was less affected by the intermittent exposures. In continuous ethylene, the colour was progressively lighter during storage after initial darkening. Shepody tubers appeared to be more sensitive to ethylene than the NorValley tubers. In Shepody only, colour in the ethylene of 1 day in 4 days treatments was progressively darker with increasing time in storage.     

Effects of Foliar and Tuber Sprout Suppressants on Storage of Ware Potatoes under Tropical Conditions

Potato (Solanum tuberosum L.) is an important source of dietary carbohydrate and cash income generation for farmers in the tropical highlands of Kenya. The feasibility for cold storage at the farm level is limited due to the high costs of maintaining such a facility and there is limited data on the long-term post-harvest storage and quality of tubers of tropical-adapted cultivars. Application of sprout suppressants to control premature sprouting of ware potato is an attractive proposition. The objectives of this study were to evaluate the efficacy of pre-harvest foliar applications of paclobutrazol (PBZ) and ethephon for sprout suppression on ware potato tubers in storage. Post-harvest spray applications of Isopropyl N-(3-chlorophenyl carbamate) chloropropham (CIPC) and 1,4-dimethylnaphthalene (DMN) on tubers as fog was also evaluated. Potato cultivars had varying levels of tuber dormancy. The tubers were stored at ambient temperature (23 C) and evaluated weekly for 24 weeks for percent of tubers sprouting, length of longest sprouts, tuber weight loss and assessed for dormancy for 24 weeks. Paclobutrazol prolonged tuber dormancy by 21–31 days and reduced tuber weight loss. Ethephon treatment had no effect on dormancy and tuber weight loss. Potato tubers treated with CIPC had greater sprout control than the other treatments in storage. Tuber response to DMN treatment varied among the three potato cultivars evaluated. The findings from this study imply that PBZ is effective in prolonging potato tuber dormancy for short-term basis at 23 C, while CIPC applied on tubers was effective for long term storage. Optimization of post-harvest potato storage can improve food security in the highland tropics.