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1.
Under EC Council Directive 2007/33/EC member states of the European Union are recommended to take soil samples ranging from 100 mL to >1500 mL to monitor populations of potato cyst nematodes [PCN] ( Globodera spp.) in targeted fields. This study examines the effects of varying sample size on sample processing and cyst extraction using two widely used laboratory methods. Cyst recovery was stable using the Fenwick can from 100 mL up to the physical limits of the can. Recovery was significantly lower for low density 50 mL samples; however, this was partly due to a statistical artefact since higher numbers of cysts were lost from successively larger samples with constant cyst numbers (i.e. decreasing densities). The Schuiling centrifuge had functional limits of <100 mL and >500 mL. Outside this range, cyst recovery from low density samples was significantly reduced. Cyst recovery from samples of varying size, but with equal numbers of cysts was only constant above 100 mL. However, samples ranging from 200–500 mL were optimal for the Schuiling centrifuge. Cyst extraction efficiency was similar using both methods for samples ranging from 150 mL to 1500 mL, where larger samples were divided into <500 mL portions. However, processing times (i.e. cyst extraction and microscopic examination) and water consumption increased significantly with increasing sample size and were significantly higher when using the Fenwick can. 相似文献
2.
J. G. N. Wander W. van den Berg P. H. J. F. van den Boogert J. G. Lamers G. C. M. van Leeuwen G. Hendrickx P. Bonants 《European journal of plant pathology / European Foundation for Plant Pathology》2007,119(2):165-174
A zonal centrifugation method, known as the Hendrickx centrifuge technique, was tested for routine detection of winter sporangia
of Synchytrium endobioticum in soil. In four experiments the ability of the Hendrickx centrifuge to extract the sporangia from soil was compared with
a method used by the Dutch Plant Protection Service, which is a modification of the recommended EPPO method. Naturally and
artificially contaminated soil samples were used to study the recovery percentage of and variation in numbers of winter sporangia.
The effects of soil type and inoculum density were studied. The Hendrickx centrifuge method, developed originally for extraction
of free living nematodes from soil, performed better than the method used by the Dutch Plant Protection Service. This was
due to a better extraction recovery (60% higher), a lower measurement error (50% lower) and a lower detection level (down
to 0.02 sporangia g−1 soil). The Hendrickx centrifuge method is much less labour-intensive than the method used by the Dutch Plant Protection Service.
It can be used to extract many different organisms from soil, and DNA can be subsequently extracted from the supernatant for
further PCR analysis. Inclusion of the Hendrickx centrifuge method in the official EPPO diagnostic protocol for regulated
pests is recommended as an alternative method for detection of sporangia in soil. 相似文献
3.
Determination of weed seeds in the soil is tedious and time consuming. To evaluate the different seed extraction methods to improve seedbank estimations, three extraction methods (sieving, cloth bag and flotation) were compared, based on their accuracy and time needed for separation processes and enumeration. Seeds of Datura stramonium,Amaranthus retroflexus,Portulaca oleracea and Plantago major were used to artificially infest soil samples of four textures, namely clay, clay loam, loam and sandy loam containing 19%, 27%, 38% and 65.5% sand respectively. Soil textures had a significant effect on counting time in all extraction methods. In flotation, four submethods involving different solutions and centrifuge rotation speed were examined, but showed no differences, so data were pooled. Counting time in flotation, cloth bag and sieving methods was 9, 16 and 30 min respectively. However, when the time needed for other processes was taken into consideration, flotation and bag methods did not differ significantly. Species‐wise seed recovery was not affected by soil texture in the bag method, suggesting an advantage for this approach as its accuracy is not soil texture‐dependent. Total seed recovery for flotation, sieving and bag methods was 61%, 67% and 75%, respectively, and was not significantly different. Considering the efficiency of methods, the cloth bag technique could be recommended, because it was as time consuming as the flotation method, but required the same minimum equipments and costs as the sieving method. 相似文献
4.
为明确贝莱斯芽胞杆菌Bacillus velezensis菌株ZF145中可有效抑制黄瓜角斑病病原菌扁桃假单胞菌流泪致病变种Pseudomonas amygdali pv.lachrymans的主要物质,采用冻干法、酸沉法、絮凝法、萃取法和铵沉法提取菌株ZF145发酵液中的抑菌物质,通过室内盆栽试验验证粗提物对黄瓜角斑病的防治效果,综合比较不同方法的提取效果,并通过高效液相色谱-质谱（high performanceliquid chromatography-mass spectrometry,HPLC-MS）联用技术鉴定粗提物中的抑菌物质。结果表明,絮凝法最适用于贝莱斯芽胞杆菌菌株ZF145发酵上清液中抑菌物质的提取,通过絮凝法提取的粗提物对扁桃假单胞菌流泪致病变种的抑菌圈直径达到19.50 mm,对黄瓜角斑病的盆栽防治效果为42.85%,且粗提物中的主要抑菌物质为C13~C16的surfactin同系物,浓度和纯度均为最高,分别为2.77 g/L和69.28%。表明本研究优化的絮凝法可用于芽胞杆菌发酵液中抑菌物质的提取。 相似文献
5.
为探索一种准确测定土壤根结线虫种群数量的方法,采用NaOCl消解附着在土壤病残体上的根结线虫卵囊与线虫常规离心技术相结合的方法,测试了不同NaOCl浓度、离心时间对根结线虫回收效率的影响,筛选出最佳测定条件,并与浅盘法、离心法、Byrd法等线虫常规分离方法进行了比较。结果表明,在9个NaOCl处理浓度中,1.0%~2.5%NaOCl处理对土壤根结线虫的回收效率显著高于3.0%NaOCl处理,达到71.6%~99.4%。在1.0%、1.5%、2.0%和2.5%NaOCl处理30 s后,分别离心2~5、2~3、2~3 min和2 min对根结线虫的回收效率明显高于其它时间处理,在每50 m L土壤平均接种976粒线虫卵的人工病土中,上述处理分离线虫数量分别达到804.3~930.0、810.7~838.7、843.7~867.0和820.7。本研究的改良方法对自然病土中根结线虫的分离数量分别是浅盘法、离心法和Byrd法的29.3、13.4和2.1倍,显著提高了土壤中根结线虫的分离效率,可准确测定土壤根结线虫种群的数量。 相似文献
6.
采用多种方法对雀麦花叶病毒德国株系(BMV-G)及英国株系(BMV-E)粒子的提纯及RNAs的提取效果进行了比较,结果分别以聚乙二醇(PEG)沉淀法及两相酚提取法为佳。聚丙烯酰胺凝胶(PAA)电泳证明BMV-G具有5个RNA组份,比BMV-E (代表典型株系)多一个RNA-3a,其分子量为0.52×106,介于RNA-3及RNA-4之间。另外变性及不变性RNAs分子量的测定结果也基本相同。薄层电泳分析指出BMV二种株系RNAs的碱基比也基本一致,同于Paul及Huth的报道。经34% CsCl平衡等密度离心得知BMV-G及BMV-E均具有4种粒子组份,其中最重的粒子的浮力比重值分别为1.3513及1.3534,而最轻的则分别达1.2993及1.3045。 相似文献
7.
Marga P. E. van Gent-Pelzer Marjon Krijger Peter J. M. Bonants 《European journal of plant pathology / European Foundation for Plant Pathology》2010,126(1):129-133
Real-time PCR was used for quantitative detection of the potato pathogen, Synchytrium endobioticum, in different substrates: zonal centrifuge extracts, warts and different plant parts of potato. Specific primers and a TaqMan
probe, designed from the internal transcribed spacer region of the multi-copy rDNA gene were tested in extracts from artificially
and naturally infested soil. Co-amplification of target DNA along with an internal competitor DNA fragment made the diagnostic
assay more reliable by guarding against false negative results. A calibrations curve was created by spiking zonal centrifuge
fractions of clean soil samples with a dilution series of winter spores. The Taqman assay was also performed on infected potato
plant material (stolons) along with the detection of the cytochrome oxidase gene as a potato endogenous control. Sensitivity
of the TaqMan assay was improved at least 100-fold and proved to be reliable for accurate diagnosis of the disease. 相似文献
8.
Xanthomonas axonopodis pv. phaseoli (Xap) is an important seedborne pathogen of Phaseolus vulgaris. Accurate seed health testing methods are critical to protect seed quality and meet phytosanitary requirements. Currently employed selective media‐based methods include several variations in extraction procedures. In order to optimize pathogen extraction from seeds, the influence of different extraction steps on the sensitivity of Xap detection was assessed. Seeds were inoculated by vacuum infiltration with Xap to achieve inoculum levels from 101 to 105 CFU per seed; one contaminated seed was mixed into 1000‐seed subsamples of uncontaminated P. vulgaris seeds. Thirty subsamples of 1000 seeds were tested using each different extraction procedure. These included soaking whole seeds in sterilized saline phosphate buffer, either overnight at 4°C or for 3 h at room temperature, with or without vacuum extraction, and either with or without concentrating the seed extract by centrifuging. Seed extract dilutions were cultured on semiselective agar media MT and XCP1. The percentages of positive subsamples were compared to measure the effects of each extraction step on detection sensitivity. Vacuum extraction and centrifugation of seed extracts increased sensitivity; the highest sensitivity was obtained with the 3 h vacuum extraction followed by centrifugation. These results were confirmed with naturally infested seeds; Xap was detected in 48 of 70 samples using the 3 h vacuum extraction with centrifugation, whereas only 35 of 70 field samples tested positive using overnight soaking, a significant difference. The results suggest that these steps would be valuable modifications to the current method approved by the International Seed Testing Association (ISTA). 相似文献
9.
PCR-based methods were developed for the detection and quantification of the potato pathogen Synchytrium endobioticum in soil extracts and in planta. PCR primers, based on the internal transcribed spacer region of the multi-copy gene rDNA were tested for specificity, sensitivity
and reproducibility in conventional and real-time PCR assays. Soil extraction procedures compared included the Hendrickx centrifugation
(HC) procedure, nested wet sieving (NWS) and a method used by the Plant Protection Service (PPS). The primers amplified a
472 bp product from S. endobioticum DNA, but did not amplify DNA from other potato pathogens, other plant pathogens, and related species. Standard cell disruption
and DNA extraction and purification methods were optimized for amplification of S. endobioticum DNA from resting sporangia. DNA was successfully amplified from a single sporangium and equivalent DNA preparations from
soil extracts. Low levels of target DNA in water did not amplify, possibly due to DNA loss during final purification steps.
A real-time PCR assay, developed for soil-based extracts using primers and probe based on the rDNA gene sequences, involved
co-amplification of target DNA along with an internal DNA fragment. Both conventional and real-time PCR methods performed
well with HC- and NWS-extracts having a threshold sensitivity of 10 sporangia per PCR assay. Of the three soil extraction
methods, only with the HC method could 100 g soil samples be efficiently processed in one single PCR assay. Such a high capacity
assay could be useful for routine soil analysis in respect to disease risk assessments and to secure de-scheduling according
to EPPO guidelines. 相似文献
10.
Leila Eshraghi Nader Aryamanesh Jonathan P. Anderson Bryan Shearer Jen A. McComb Giles E. St. J. Hardy Philip A. O’Brien 《European journal of plant pathology / European Foundation for Plant Pathology》2011,131(3):419-430
A reliable method for measuring disease progression is important when evaluating susceptibility in host—pathogen interactions.
We describe a sensitive quantitative polymerase chain reaction (QPCR) assay that enables quantitative measurement of in planta DNA of the necrotrophic pathogen, Phytophthora cinnamomi, that avoids problems caused by variation in DNA extraction efficiency and degradation of host DNA during host tissue necrosis.
Normalization of pathogen DNA to sample fresh weight or host DNA in samples with varying degrees of necrosis led to overestimation
of pathogen biomass. Purified plasmid DNA, containing the pScFvB1 mouse gene, was added during DNA extraction and pathogen
biomass was normalized based on plasmid DNA rather than host DNA or sample fresh weight. This method is robust and improves
the accuracy of pathogen measurement in both resistant (non-host A. thaliana–P. cinnamomi) and susceptible (host Lupinus angustifolius–P. cinnamomi) interactions to allow accurate measurement of pathogen biomass even in the presence of substantial host cell necrosis. 相似文献
11.
超高效液相色谱-串联质谱法同时检测大米中15种常用农药的残留 总被引：3，自引：1，他引：2
建立一种超高效液相色谱-串联质谱同时检测大米中甲氨基阿维菌素苯甲酸盐等15种常用农药残留的方法,考察了基质效应、提取溶剂种类、提取方法以及不同净化方法对15种农药回收率的影响。样品经V (乙腈) : V (丙酮) : V (水) = 16 : 2 : 2混合溶液匀浆提取,分散固相萃取法净化,电喷雾正离子 (ESI+) 模式电离,多反应监测 (MRM) 模式检测,外标法定量。结果表明:在0.005~1 mg/L范围内,甲氨基阿维菌素苯甲酸盐等15种农药的质量浓度与对应的峰面积间线性关系良好,相关系数 (r) ≥ 0.99;在0.01、0.1、1和4 mg/kg 4个添加水平下,15种农药在大米中的平均回收率在82%~116%之间,相对标准偏差在1.2%~12%之间 (n = 5)。方法检出限为0.000 5~0.005 mg/kg,定量限为0.01 mg/kg。用该方法对上海市郊20个批次的大米样品进行测定,均未检出农药残留超标。该方法操作简单、快速、准确,适用于大米中甲氨基阿维菌素苯甲酸盐等15种常用农药残留的同时检测。 相似文献
12.
建立了蔬菜中烯肟菌胺残留量的固相萃取-气相色谱-质谱联用（SPE-GC-MS）分析方法;对烯肟菌胺的裂解机理进行了探讨;确定了测试分析的定性离子和定量离子。样品采用乙酸乙酯高速分散提取,以无水硫酸镁、硅胶和石墨化炭黑（GCB）为混合填料进行固相萃取净化,气相色谱-质谱联用仪在选择离子扫描（SIM）模式下进行检测,基质匹配标准溶液外标法定量。结果显示:烯肟菌胺在0.02~1 mg/L内,标准溶液的峰面积与质量浓度呈良好的线性关系（r>0.999）;在0.02~0.2 mg/kg添加水平下,烯肟菌胺的平均回收率为94%~99%,相对标准偏差（RSD,n=6）为2.3%~2.9%;方法的检出限（LOD）（S/N=3）为0.006 mg/kg,定量限（LOQ）（S/N=10）为0.02 mg/kg。 相似文献
13.
放线菌菌株MY02的鉴定及发酵液中抗真菌活性组分的分离 总被引：1，自引：0，他引：1
从东北地区蔬菜保护地土壤中筛选分离到一株放线菌菌株MY02,通过对其形态学特征及16S rDNA序列分析,初步鉴定其为龟裂链霉菌龟裂亚种Streptomyces rimosussub sp.rimosus。该菌株的发酵液具有抗真菌活性。发酵液经离心、过滤、萃取、硅胶柱层析等步骤分离纯化其活性物质,并进行高效液相色谱(HPLC)分析,表明其活性物质中含有两种抗真菌活性组分。对活性组分的正丁醇溶液进行紫外光谱扫描,结果表明该活性组分具有四烯类抗生素的典型特征吸收峰。 相似文献
14.
15.
A simple extraction method with water is described which allows the quantitative determination, within 1 day of the residues of soil-applied herbicides which are potentially available to plants. The validity of this method has been examined with a growth test using Brassica rapa in soils containing residues of different ages. 相似文献
16.
Naruto FURUYA Hiroyuki URA Kazuhiro IIYAMA Masaru MATSUMOTO Minoru TAKESHITA Yoichi TAKANAMI 《Journal of General Plant Pathology》2002,68(3):220-224
Specific primers were designed based on the sequences of the spacer region between the 16S and 23S ribosomal DNA (rDNA) for
direct, rapid and specific detection of Burkholderia gladioli. These primers were named GLA-f and GLA-r. PCR performed on boiled bacterial suspensions yielded an amplification product of
approximately 300 bp. No products from other bacterial species, including B. glumae were amplified, even after complete DNA extraction by the cetyltrimethyl-ammonium bromide (CTAB) method. Using the specific
primers designed in this study, the PCR method can detect B. gladioli in plant samples within 6 hr. These data demonstrate the potential of specific PCR for the detection of B. gladioli.
Received 10 December 2001/ Accepted in revised form 15 April 2002 相似文献
17.
优化的QuEChERS-超高效液相色谱-串联质谱法检测黄瓜中29种杀菌剂残留 总被引：1，自引：1，他引：0
建立了采用QuEChERS-超高效液相色谱-串联质谱（UPLC-MS/MS）同时检测黄瓜中29种杀菌剂残留的分析方法。黄瓜样品经乙腈提取和前处理方法优化,以乙二胺-N-丙基硅烷（PSA）和石墨化碳黑（GCB）为分散固相萃取（dSPE）吸附剂,涡旋提取后过滤膜,采用UPLC-MS/MS检测。结果表明:在0.000 1~0.05 mg/kg范围内,29种杀菌剂的质量浓度与相应的峰面积间呈良好的线性关系,相关系数均大于0.996,定量限（LOQ）在0.000 1~0.01 mg/kg之间;在0.01、0.05和0.5 mg/kg添加水平下,29种杀菌剂的平均回收率均在74%~112%之间,相对标准偏差（RSD）在1.1%~17%之间（n=5）。经实际样品检测验证,该方法快速、简便、可靠、高效,且成本低廉,适用于同时检测黄瓜中29种杀菌剂的残留量。 相似文献
18.
Xuefeng Wang Changyong Zhou Kezhi Tang Yan Zhou Zhongan Li 《European journal of plant pathology / European Foundation for Plant Pathology》2009,124(1):175-180
Citrus plants are natural hosts of five viroid species and large numbers of sequence variants. In this paper a simple and
sensitive one step multiplex RT-PCR protocol with an internal control was utilised to simultaneously detect and differentiate
five citrus viroids: Citrus exocortis viroid (CEVd), Citrus bent leaf viroid (CBLVd), Hop stunt viroid (HSVd), Citrus viroid-III (CVd-III) and Citrus viroid-IV (CVd-IV). In addition, a micro and rapid total nucleic acid extraction method was developed and the protocol applied to evaluate
the occurrence and distribution of citrus viroids in China. 相似文献
19.
P. Parikka A. Lemmetty 《European journal of plant pathology / European Foundation for Plant Pathology》2004,110(4):393-398
Colletotrichum acutatum, a quarantine organism on strawberries in the EU, was found in Finland for the first time in 2000. Concern about rapid, unnoticeable spread of this pathogen has necessitated studies to find methods with which the quiescent fungus infection can be detected in imported, cold-stored strawberry plant material. Successful detection of C. acutatum in strawberry tissues by polymerase chain reaction (PCR) is dependent on the method of DNA extraction used. Good-quality nucleic acid, free of PCR inhibitors, was successfully prepared by slightly modifying the DNA extraction method of a commercially available kit. Species-specific primers, previously described in the literature, were successfully used in the PCR reaction. C. acutatum was detected by PCR both on symptomatic and asymptomatic plant parts and in artificially and naturally infected strawberry tissues. Positive PCR results were obtained from ripe and unripe berries, runners, petioles and different parts of crowns. The data demonstrate that the PCR technique can be used to detect C. acutatum in strawberry tissue even in plant parts that do not show visible symptoms. 相似文献
20.
建立了黄瓜和番茄中毒氟磷残留量的固相萃取-气相色谱-质谱联用(SPE-GC-MS)分析方法;对毒氟磷的裂解机理进行了探讨,确定了测试分析的定性离子和定量离子。样品采用V(正己烷):V(丙酮)=1:1混合液高速匀浆提取,使用以N-丙基乙二胺(PSA)、C18和石墨化炭黑(GCB)的混合物为填料的固相萃取柱净化,采用气相色谱-质谱联用仪在选择离子扫描(SIM)模式下进行检测,外标法定量。结果显示,毒氟磷在0.01~0.5 mg/L内,标准溶液的峰面积与质量浓度的线性关系良好(r≥0.999),方法的检出限(LOD)(S/N=3)为0.003 mg/kg,定量限(LOQ)(S/N=10)为0.01 mg/kg。在0.01、0.02和0.1 mg/kg添加水平下,毒氟磷的平均回收率为99.9%~100.2%,相对标准偏差(RSD,n=6)为0.97%~3.07%。 相似文献