梨火疫病菌的实时荧光PCR检测

 根据梨火疫病菌16S~23S间的ITS保守序列,设计并合成了一对特异性引物REA/FEA,应用荧光染料SYBR Green I,对10个梨火疫的菌株和其它相关参试菌株进行了检测。结果表明,10个梨火疫菌株都产生荧光信号而其它参试菌株都不产生荧光信号,成功建立了梨火疫病菌的实时荧光PCR检测方法。整个检测过程只需3h,完全闭管,降低了污染的机会,无需PCR后处理。检测的灵敏度是4个菌体细胞,比常规PCR电泳检测提高了10倍。用该特异性引物对梨枝条浸泡液进行实时荧光PCR检测,结果可特异性检测到目标菌的存在,并且检测的灵敏度是24个菌体细胞,比常规PCR电泳检测提高10倍。     

丁香疫霉病菌PCR和实时荧光PCR检测

 为了快速、准确地检测丁香疫霉病菌 (Phytophthora syringae, PSY),根据GeneBank中PSY的ITS序列设计特异引物Psy1/Psy2和探针P-Psy,建立了常规PCR和实时荧光PCR检测方法。利用引物Psy1/Psy2扩增供试的26株PSY能得到585 bp的预期目标条带,但扩增其它61个非PSY供试菌株不能得到预期产物,检测灵敏度为12 pg菌丝DNA;探针P-Psy对供试26株PSY表现为阳性扩增,而对其它菌株和空白对照均表现为阴性扩增,检测灵敏度可达120 fg菌丝DNA,比常规PCR高100倍;引物Psy1/Psy2和探针P-Psy对5 g土壤中PSY卵孢子的检测灵敏度分别为20 000个和200个。样品检测试验表明两种PCR方法可用于口岸植物检疫中快速、准确和特异地检测丁香疫霉病菌。     

杂交诱捕实时荧光PCR检测番茄环斑病毒

 番茄环斑病毒(Tomato ringspot nepovirus,ToRSV)广泛分布于欧美及大洋洲,能侵染葡萄、桃、苹果、樱桃、番茄及烟草等150多种植物,引起多种病害,严重时导致绝产^[1]。     

美国进境樱桃果实中梨火疫病菌的检测

为了筛选快速、灵敏的梨火疫病菌检测方法,利用常规PCR、套式PCR和实时荧光PCR方法分别对美国进境的326批樱桃果实中梨火疫病菌进行检测。结果显示,3种PCR方法的检出率不同,不同引物或探针的检出率也存在差异。在常规PCR中,引物Ams3/Ams4c、P29A/P29B和PEANT1/PEANT2的检出率分别为35.28%、24.85%和16.87%;单管套式PCR和套式PCR的检出率分别为23.01%和50.61%;4种实时荧光PCR的检出率分别为17.48%(探针PA)、32.21%(探针Ams)、29.14%(探针ITS)和23.93%(SYBR GreenⅠ)。在所有试验方法中由引物P29A/P29B和PEANT1/PEANT2组成的套式PCR的检出率最高。检测结果证实了进境樱桃果实中存在梨火疫病菌DNA,套式PCR和常规PCR(引物Ams3/4c)可用于进境樱桃样品中梨火疫病菌的常规检测。     

梨衰退植原体Cycleave 实时荧光PCR检测方法的建立

 根据植原体16S rDNA 保守区设计Cycling 探针LST2probe及引物,建立了梨衰退植原体Cycleave实时荧光PCR检测方法。结果表明,探针LST2probe 能特异的检测梨衰退植原体,供试同一组内不同亚组植原体及参试病原细菌均为阴性,检测灵敏度可达0.5 pg/μL。该Cycleave实时荧光PCR检测方法可用于梨衰退植原体的快速检测,并为其他有害生物鉴定提供借鉴依据。     

检测梨火疫病菌的过敏性寄主

梨火疫病（ＦｉｒｅＢｌｉｇｈｔ）是梨、苹果及许多蔷薇科植物上一种毁灭性的细菌病害,也是我国进境植物应检疫的危险性病害之一。植物病原假单胞菌的过敏反应测定通常在烟草和蕃茄上进行,稻白叶枯病菌的过敏性反应测定还可在蚕豆叶上进行［１］,１９７３年Ｌａｉ和Ｈ...     

冬生疫霉的实时荧光PCR检测方法

冬生疫霉(Phytophthora hibernalis)是我国检疫性病原菌。本研究根据冬生疫霉的ITS基因序列,设计了实时荧光PCR引物PH-F和PH-R及TaqMan-MGB探针PH-Pr,建立了冬生疫霉的实时荧光PCR检测方法,可检测到冬生疫霉DNA最低浓度为50 fg/μL,而冬生疫霉的近似种、近缘种和空白对照,无荧光信号增加。因此,利用该方法可以稳定、高效的检测出冬生疫霉。     

免疫捕获PCR检测进境苹果果实中梨火疫病菌

利用梨火疫病菌抗体和PCR技术建立了梨火疫病菌免疫捕获PCR方法,检测苹果模拟样品的灵敏度达到了1.5×102cfu/reaction。与直接PCR进行比较,免疫捕获PCR方法检测苹果模拟样品的灵敏度提高了10倍以上,而且省去了DNA提取等步骤。利用该方法成功从进境苹果样品中检测到梨火疫病菌,产物测序结果表明,产物序列和梨火疫病菌的相应序列高度一致。试验结果表明,免疫捕获PCR法能除去样品中的大部分PCR反应抑制物质,可以有效检测进境苹果中梨火疫病菌,在口岸水果检疫中具有一定的应用潜力和推广价值。     

进境苹果果实中梨火疫病菌的套式PCR检测

 针对进境商用苹果果实携带梨火疫病菌Erwinia amylovora数量有限的特点,选取源于病菌pEA29质粒的2对引物P29A/P29B和PEANT1/PEANT2配对组合成套式PCR,其检测灵敏度可达0.15 pg菌体DNA,检测灵敏度高于EPPO推荐的单管套式PCR方法和常规PCR方法。分别利用这3种PCR检测方法对美国、新西兰、日本和智利等国进境的166批苹果样品进行检测,3种检测方法的样品阳性率分别为53.6%、38.0%和8.4%,试验结果表明此套式PCR检测方法可用于进境商用苹果的梨火疫病菌快速检测。进境样品的检测结果证实了进境商用苹果果实中存在梨火疫病菌的可能性。     

ELISA及实时荧光PCR检测番茄细菌性溃疡病菌的方法比较

本文以番茄细菌性溃疡病菌(Clavibacter michiganensis subsp.michiganensis)菌悬液和田间采集的病组织为材料,比较ELISA试剂金、常规PCR方法和实时荧光PCR方法检测番茄细菌性溃疡病菌灵敏度和适用性.结果表明,ELISA试剂盒检测灵敏度为105cfu/mL,具有简便、快速、易操作特点,适用于田间病害诊断;常规PCR检测灵敏度为105cfu/mL;TaqMan探针实时荧光PCR检测灵敏度为103～4cfu/mL,比常规PCR和ELISA检测灵敏度提高10～100倍,且不需要琼脂糖凝胶电泳,溴化乙锭染色和Southem杂交,但需要昂贵的仪器和试剂,适用于室内检测及相关研究.     

Improved fireblight diagnostics using quantitative real-time PCR detection of Erwinia amylovora chromosomal DNA

Specific and sensitive TaqMan real-time PCR assays were developed targeting chromosomal DNA of Erwinia amylovora ( ams C gene and ITS region). These assays increased the reliability of detection of E. amylovora strains, regardless of their plasmid profile, and have the ability to differentiate between Erwinia spp. strains from Hokkaido, Erwinia pyrifoliae and Erwinia spp. isolated from necrotic pear blossoms in Spain. The assays were used for testing the efficiency of three different extraction methods to remove plant-based PCR inhibitors. Combined with an automated DNA-extraction method based on magnetic beads (QuickPick™), the real-time PCR assays reliably detected at least 10³ cells mL^−l ( c. four cells per reaction) of the pathogen from blighted woody plant material. In testing of symptomless samples, absolute quantification of E. amylovora before and after enrichment in liquid media provided proof of E. amylovora viability and its ability to multiply, including in cases when subsequent isolation in pure culture was unsuccessful.     

梨火疫病的分布,传播及检测技术研究进展

本文介绍了梨火疫病的危害,传播及目前世界分布;讨论了各种植物材料和包装物传病的危害性,评价了包括传统的和分子生物学方法在内的梨火疫病菌的各种检测方法及该病对我国的潜在威胁,为对该病的检疫检测提供理论依据。     

梨火疫病菌的RPA快速检测技术的研发

梨火疫病菌Erwinia amylovora是我国进境植物检疫性有害生物, 主要侵染梨、苹果、山楂、海棠等多种蔷薇科植物引起梨火疫病。该病自1780年在美国被发现以来, 已经传播到世界多个国家, 对我国的果树生产也构成一定的威胁。严格植物检疫是全球防控梨火疫病菌扩散危害的重要方法之一, 目前我国植物检疫部门主要从实验室分离鉴定、分子检测和田间症状识别及快速检测方面开展工作。本研究基于重组酶聚合酶扩增(RPA)技术和E.amylovora 16S-23S ITS区间的保守序列设计的引物和探针, 建立了实时荧光RPA和侧流层析试纸条RPA两种检测方法。经测试, 在39℃条件下, 这两种检测方法都具有良好的特异性, 分别能在20 min和10 min内完成扩增。实时荧光RPA对E. amylovora菌株DNA的检测阈值为1.38×10-2 ng/μL;侧流层析试纸条RPA对E. amylovora菌悬液和DNA的检测阈值分别为104 cfu/mL和1.38×10-3 ng/μL。侧流层析试纸条RPA体系因反应快速、读取结果方便、不需要复杂仪器操作可以满足现场快速检测的需求。     

梨火疫病菌对噻唑锌及其他几种杀细菌剂的敏感性

噻唑锌是近期在我国登记为防治梨火疫病的新型杀菌剂。为明确梨火疫病菌Erwinia amylovora对噻唑锌及其他几种杀细菌剂的敏感性现状,本研究选用2016年-2020年从新疆不同地区分离的50个E. amylovora 代表菌株,通过药剂-病原菌共培养结合平板菌落法测定E. amylovora 对噻唑锌的敏感性,比较不同来源菌株的敏感性差异,并分析E. amylovora 对噻唑锌与农用链霉素、噻霉酮和春雷霉素的敏感性差异。结果表明,来自新疆不同地区、不同寄主和不同年份的E. amylovora 菌株对噻唑锌的敏感性差异明显,EC50分布在2.67~38.13 μg/mL, 平均（18.60±6.35） μg/mL。阿克苏地区阿瓦提县的杜梨分离菌株H10最敏感,EC50为2.67 μg/mL,巴州轮台县的山楂分离菌株Y126的敏感性最低,EC50为38.13 μg/mL,二者的EC50相差13.28倍。供试E. amylovora 菌株对噻唑锌的敏感性频率分布呈连续的单峰曲线,符合正态分布,将EC50平均值(18.60±6.35)μg/mL确定为新疆梨火疫病菌对噻唑锌的敏感基线,适用于新疆梨火疫病菌对噻唑锌的抗药性检测。供试E. amylovora 菌株对噻唑锌的抗性水平为0.13~2.05 (均值为1.00±0.35),均为敏感菌株,未发现抗药性菌株。相关性分析表明,E. amylovora 对噻唑锌的敏感性与农用链霉素（r=0.138,P=1.56）、噻霉酮(r=0.417,P=0.78)和春雷霉素(r=0.434,P=0.65)的敏感性之间不存在相关性(P>0.05)。本研究结果为后期评估梨火疫病菌对噻唑锌的抗药性风险奠定了基础,为制定梨火疫病的合理用药策略提供了依据。     

Identification by PCR analysis on plasmid pEA29 of isolates of Erwinia amylovora responsible of an outbreak in Central Europe

A collection of 127 strains of Erwinia amylovora, the causative agent of fire blight, was tested by PCR amplification of a fragment of the plasmid pEA29. A variability in the length of the DNA fragment obtained was observed after digestion by MspI and Sau3A restriction enzymes. Strains were distributed into three groups according to the length of the DNA product. Most of the strains analysed were placed into two groups. Thirteen strains were clustered into a third group which was linked with the geographical origin of strains: they were all isolates from recently reported outbreaks of fire blight in Austria and in southern Bavaria in Germany. The variation in the length of the amplified fragment is probably due to an insertion into this fragment.     

Real‐time PCR detection of Erwinia amylovora on blossoms correlates with subsequent fire blight incidence

Fire blight is the most devastating bacterial disease of rosaceous plants. Forecasting fire blight infections is important to allow for countermeasures that reduce economic damage in pome fruit production. Current computerized forecasting models are solely based on physical factors such as temperature and moisture, but not on the actual presence of the pathogen Erwinia amylovora. Although the inoculum concentration is considered to be crucial for infection and disease outbreak, most current approaches used for identification of fire blight inoculum including morphological, biochemical, serological, and DNA‐based methods are nonquantitative. Based on a real‐time PCR approach previously published, an improved protocol to be used directly on whole bacteria in the field is described. The method allows for early detection and quantification of the pathogen prior to the occurrence of first symptoms. There is a clear correlation between bacterial abundance and subsequent disease development. However, in some cases, no disease symptoms could be observed despite a pathogen load of up to 3·4 × 10⁶ cells per blossom. Integration of the amount of pathogen detected into refined prediction algorithms may allow for the improvement of applied forecasting models, finally permitting a better abatement of fire blight.     

Distribution of streptomycin-resistant strainsof Erwinia amylovora in Israel and occurrence of blossom blight in the autumn

Following failure in control of fire blight with streptomycin, the distribution of streptomycin-resistant strains ofErwinia amylovora in Israel was surveyed. During 1994–1997 109 pear, apple, loquat and quince orchards were monitored. Streptomycin-resistant strains ofE. amylovora were recovered from flowers and from infected branches collected at 18 locations in the Sharon, Galilee and Golan Heights regions. In the Sharon region all the isolated strains ofE. amylovora were streptomycin-resistant, whereas in the Galilee and Golan Heights, resistant as well as sensitiveE. amylovora strains were recovered at different locations. In the southern coastal plain no resistance could be detected. Streptomycin-resistant strains ofE. amylovora did not hybridize with the DNA probe SMP3, and resistance could not be transferred by mating to a sensitive strain, suggesting that streptomycin resistance in Israel is not plasmid-mediated. Fire blight symptoms were observed, for the first time, on pear blossoms during the autumn of 1994. A high population of 2x 10⁶-6x 10⁷ CFU/flower in the autumn of 1995 and of 1996 was correlated with the appearance of blossom blight symptoms.     

长孢轮枝菌Taq〖KG-*4〗Man-MGB探针实时荧光PCR快速检测方法

长孢轮枝菌是一种在我国局部地区新近出现且危害性极大的植物病原真菌?根据长孢轮枝菌及其近似种的actin序列差异, 设计并合成特异性引物和探针, 建立了长孢轮枝菌的实时荧光PCR检测方法?特异性试验结果表明, 该检测方法能特异性检测长孢轮枝菌; 灵敏度试验结果表明, 最低检测限量为10 μL反应体系中总DNA含量10 pg; 实时荧光PCR优化反应条件为引物终浓度0.8 μmol/L, 探针终浓度0.8 μmol/L, 优化后的整个反应过程约1 h?实际样品检测结果表明, 该方法可用于疑似受长孢轮枝菌侵染的萝卜样品检测与初筛?此方法快速?灵敏, 检测过程完全闭管, 无需PCR后续处理, 为早期快速检测长孢轮枝菌提供了重要参考?     

应用实时荧光定量技术评价粉红粘帚霉对水稻纹枯病的田间防治效果

 本研究利用实时荧光定量PCR技术监测施用生防菌前后土壤中粉红粘帚霉67-1和水稻纹枯病菌AG-1IA的数量,并调查发病情况,收割后测产,评估生防菌粉红粘帚霉67-1的田间防治效果。实时荧光定量检测结果表明,粉红粘帚霉67-1防治水稻纹枯病的效果随着用药浓度的增加而增强,各处理间差异极显著。用药后土壤中粉红粘帚霉67-1的含量在一定时间内随培养时间的延长逐渐升高,停止用药后逐渐减少。水稻纹枯病菌在清水对照组中没有显著变化,在处理组中水稻纹枯病菌的数量随时间降低。60 g/667 m²菌量的防效及产量与井冈霉素对照组差异不显著。防病保产结果与实时荧光定量结果基本一致,说明粉红粘帚霉67-1的孢子制剂对水稻纹枯病具有优良的田间防治效果,实时荧光定量PCR技术可以用于生物防治效果的田间评价。     

Characterisation of naturally occurring <Emphasis Type="Italic">Erwinia amylovora</Emphasis> strains lacking the common plasmid pEA29 and their detection with real-time PCR

Erwinia amylovora, the causal agent of fire blight, carries the common plasmid pEA29 of 29 kb. To screen for occurrence of natural strains without plasmid pEA29, we applied PCR analysis with primers from the plasmid and the chromosomal ams region. In addition, a described TaqMan probe from pEA29 and newly designed primers from the ams-region were used for identification by real-time PCR. One strain isolated in Iran, one strain from Spain and two strains from Egypt lacked plasmid pEA29. From a recent screening series in southern Germany, in 123 E. amylovora strains from necrotic fire blight host plants, one strain was found without the common plasmid. The strains without pEA29 were virulent in assays with immature pears and on apple seedlings, but showed a reduced growth level in minimal medium without amino acids and thiamine. Transposon-labelled pEA29 was transformed into the plasmid-free strains resulting in restoration of this growth deficiency. The plasmid was stably maintained in these E. amylovora cells. The newly designed chromosomal primers for conventional and for real-time PCR identified E. amylovora strains in field samples lacking pEA29. These variants are apparently rare, but were detected in isolates from different regions in the world with fire blight.