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1.
油菜茎基溃疡病菌LAMP-LFD检测方法的建立   总被引:1,自引:0,他引:1  
本文基于环介导等温扩增技术与横向流动试纸条相结合的方法,建立了一种应用于油菜茎基溃疡病菌(Leptosphaeria maculans)的LAMP-LFD快速检测方法。以油菜茎基溃疡病菌的ITS基因序列为靶序列,设计出一套用于LAMP-LFD检测的引物和探针,优化了反应体系与反应条件(63℃,35 min)。结果表明:只有油菜茎基溃疡病菌出现阳性条带,其他参照菌株和阴性对照均未出现阳性条带,说明LAMP-LFD检测特异性强;灵敏度检测表明,对油菜茎基溃疡病菌的检测极限可低至114 fg/μL,灵敏度比传统PCR高10倍;该方法可从进境船载油菜籽样品中成功检测出油菜茎基溃疡病菌,检测结果与传统的鉴定方法一致。LAMP-LFD检测方法能够快速检测油菜茎基溃疡病菌,具有简便、灵敏、特异性高,不依赖特殊检测设备等优点,极具推广前景。  相似文献   

2.
进境油菜籽中黑胫病菌和茎基溃疡病菌的检测   总被引:1,自引:0,他引:1  
为准确鉴定从进境澳大利亚油菜籽样品中分离的真菌分离物,利用形态学特征、PCR检测、序列分析以及致病性测试等方法对分离物6382-43和6382-51进行了鉴定试验。结果表明,分离物6382-43的形态特征和油菜茎基溃疡病菌Leptosphaeria maculans相似,菌丝生长较慢,菌落边缘不规则,不产生色素。油菜茎基溃疡病菌特异性引物LmacF/LmacR检测为PCR阳性;ITS区序列和油菜茎基溃疡病菌的序列相似性为99.8%;接种幼嫩油菜子叶产生油菜茎基溃疡病的典型症状。分离物6382-51的形态特征和油菜黑胫病菌L.biglobosa相似,菌丝生长较快,菌落边缘规则,产生色素;油菜黑胫病菌特异性引物LbigF/LmacR检测为PCR阳性;ITS区序列和油菜黑胫病菌的序列相似性为100%;接种幼嫩油菜子叶产生油菜黑胫病的典型症状。根据分离物的形态特征、PCR检测结果、序列分析以及致病性测试结果,将进境澳大利亚油菜籽样品中的真菌分离物6382-43和6382-51分别鉴定为油菜茎基溃疡病菌Leptosphaeria maculans和油菜黑胫病菌L.biglobosa。  相似文献   

3.
采用常规平板分离法, 从进境澳大利亚大麦中夹杂的油菜籽上获得1株疑似油菜茎基溃疡病菌的菌株01829?通过致病性测定?形态学观察?特异性引物扩增?ITS序列比对分析, 对01829进行了种类鉴定?结果表明:菌株01829在PDA培养基上生长较慢, 菌落边缘不整齐, 产生大量分生孢子器和分生孢子; 采用特异性引物对LMR1-D和Lmb分别进行PCR检测, 结果均有预期扩增片段产生; 基于ITS序列构建的系统发育树中, 菌株01829和GenBank中其他油菜茎基溃疡病菌相关序列聚在同一分支; 菌株01829接种油菜子叶和茎基部, 在子叶和茎基部接种部位分别引起叶斑和凹陷溃疡斑?根据上述试验结果, 将菌株01829鉴定为油菜茎基溃疡病菌, 这是我国口岸首次从进境澳大利亚大麦中截获油菜茎基溃疡病菌?  相似文献   

4.
从加拿大进境油菜籽中随机选取船载油菜籽、筒仓油菜籽、菜籽粕和下脚料4类加工过程的样品调查油菜茎基溃疡病菌的发生情况。结果表明,18份船载油菜籽、7份筒仓油菜籽和11份下脚料样品的油菜茎基溃疡病菌PCR检测都呈阳性,而8份菜籽粕样品全为阴性。3份阳性样品的病原菌分离、鉴定与致病性测试结果证实了PCR的检测结果。  相似文献   

5.
比较磁珠法、离心柱法、CTAB法及快速DNA裂解检测试剂对油菜籽中油菜茎基溃疡病菌的检测结果,综合考虑各提取方法的效率及成本。结果显示离心柱法性价比最高,快速DNA裂解检测试剂对油菜籽中油菜茎基溃疡病菌快速筛查具有更佳的应用价值。  相似文献   

6.
本研究根据向日葵白锈病菌大亚基核糖体RNA基因序列,向日葵黑茎病菌的ITS-5.8S r RNA基因序列,分别设计特异性DPO(dual priming oligonucleotide)引物,建立同时检测这两种检疫性病菌的多重DPO-PCR检测方法,并对其特异性和灵敏度进行评价。结果表明,所设计的DPO引物特异性强,仅向日葵白锈病菌和向日葵黑茎病菌可分别扩增出307 bp与388 bp的特异性条带,其他参照菌株及阴性对照均无条带;检测体系对混合模板中向日葵白锈病菌和向日葵黑茎病菌的DNA灵敏度均达0.05 ng/μL;且该检测方法对退火温度不敏感,适用范围广。该方法能够准确、快速的检测向日葵白锈病菌和向日葵黑茎病菌,适合于口岸实验室的快速检测。  相似文献   

7.
 向日葵黑茎病菌是我国进境检疫性有害生物名录中的一种检疫性真菌。根据向日葵黑茎病菌及其近似种的ITS序列差异,设计并合成特异性引物和探针,建立了向日葵黑茎病菌的实时荧光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向日葵黑茎病菌;灵敏度试验结果表明,最低检测限量为20 μL反应体系中总DNA含量0.1 pg;实时荧光PCR优化反应条件为引物终浓度0.6 μmol·L-1,探针终浓度0.3 μmol·L-1。实际样品检测结果表明,该方法可用于疑似携带向日葵黑茎病菌样品的检测与初筛。此方法快速、灵敏,整个反应过程约1 h,检测过程完全闭管,无需PCR后续处理,为早期快速检测向日葵黑茎病菌提供了重要参考。  相似文献   

8.
利用实时荧光定量PCR 技术检测油菜菌核病菌   总被引:2,自引:0,他引:2  
 采用高通量实时荧光定量PCR 技术建立检测和监测油菜菌核病菌群体数量的方法。利用油菜菌核病菌(Sclerotiniasclerotiorum)的茁-微管蛋白基因内含子序列的特异性,设计引物对SclSF (5'-CTCAAATCTCCGAAAGTT -3') / SclAF (5'-TGCAGACGGGTAATATG -3'),建立和优化了SYBR Green 玉实时荧光定量PCR 检测体系。结果表明,该引物对能够从8种所测试的十字花科植物常见病原真菌中特异性扩增出油菜菌核病菌;所建立的实时定量PCR 技术可应用于油菜病叶和病茎中菌核病菌的早期检测及菌核病的预测预报。  相似文献   

9.
为准确快速筛查进境油葵种子中携带的向日葵黑茎病菌Plenodomus lindquistii,采用常规PCR方法比较了3对向日葵黑茎病菌PCR检测引物320FOR/320RVE、LEPB/LEPF和LLF/LLR的灵敏度,对哈萨克斯坦进境120批油葵种子样品进行PCR检测,并对检测为阳性的样品进行产物序列测定及病原菌分离。结果显示,引物320FOR/320RVE的检测灵敏度最高,可达10-5ng/μL,引物LEPB/LEPF和LLF/LLR分别为10-4ng/μL和10-3ng/μL。利用引物320FOR/320RVE、LEPB/LEPF和LLF/LLR对120批进境油葵种子样品进行检测,阳性检出率分别为65%、50%和45%,阳性检出率高低与引物的检测灵敏度相一致。阳性样品扩增的产物序列与Gen Bank中向日葵黑茎病菌的序列同源性最高;分离的病原菌菌落为乳白色至灰白色,分生孢子器黑褐色、球形并产生透明状分生孢子液,分生孢子单胞、无色、肾型、两端有油球,与已报道的向日葵黑茎病菌的病原特征描述一致,初步鉴定哈萨克斯坦进境油葵种子中存在向日葵黑茎病菌。  相似文献   

10.
采用常规检测法、免疫斑点法、酶联免疫检测法和PCR检测法对柑橘溃疡病菌纯培养和柑橘叶片提取液进行平行测定。结果表明,对柑橘叶片提取液的检测,4种检测法检出的样品带菌率分别为11.12%、27.22%、18.69%、20.31%;对柑橘溃疡病菌纯培养的检测,4种方法检测结果一致。本实验同时比较了几种方法的灵敏度、特异性和稳定性,以及不同方法在各级检验检疫中的适应性。  相似文献   

11.
The potential use of DNA-based methods for detecting airborne inoculum of Leptosphaeria maculans and Pyrenopeziza brassicae , both damaging pathogens of oilseed rape, was investigated. A method for purifying DNA from spores collected using Hirst-type spore samplers and detecting it using polymerase chain reaction (PCR) assays is described. For both pathogens, the sensitivities of the DNA assays were similar for spore-trap samples and pure spore suspensions. As few as 10 spores of L. maculans or P. brassicae could be detected by PCR and spores of both species could be detected against a background of spores of six other species. The method successfully detected spores of P. brassicae collected using spore traps in oilseed rape crops that were infected with P. brassicae. Leptosphaeria maculans spores were detected using spore traps on open ground close to L. maculans -infected oilseed rape stems. The potential use of PCR detection of airborne inoculum in forecasting the diseases caused by these pathogens is discussed.  相似文献   

12.
In June/July 2001, 2002, 2003 and 2006, regional variation in distribution of the pathogens Leptosphaeria maculans and L. biglobosa that are causally associated with phoma stem canker was surveyed on winter oilseed rape crops in England. In 2001–2003, when isolates from basal cankers were visually identified as L. maculans or L. biglobosa based on cultural morphological characteristics, 70% were L. maculans and 30% L. biglobosa . In 2001, 2002, 2003 and 2006, when amounts of DNA of each species in basal cankers were determined by quantitative PCR, the abundance of L. maculans DNA was greater than that of L. biglobosa DNA in 77% of samples. When regional differences in amounts of L. maculans and L. biglobosa DNA were mapped geostatistically, quantities of L. maculans DNA were greater in cankers from southern England and those of L. biglobosa DNA were greater in northern England. A comparison with geostatistically mapped predictions made using a weather-based model describing stages in development of phoma stem canker epidemics suggested that these differences in Leptosphaeria populations may have been a consequence of differences in temperature after onset of leaf spotting between northern and southern England. Both PCR and morphological evidence suggested that the abundance of L. maculans in England has increased since the last surveys in the 1980s. Implications of these surveys for control of phoma stem canker are discussed.  相似文献   

13.
 根据油菜茎基溃疡病菌Leptosphaeria maculans与其近似种ITS序列的差异,设计了检测L. maculans的引物Lmb3/R2和探针Probe-M,建立了L. maculans的实时荧光PCR检测方法。试验结果表明,来自加拿大、澳大利亚和乌克兰等国的22株L. maculans菌株都能得到阳性扩增,而供试的30株L. biglobosa菌株和6株其他菌株以及空白对照没有荧光信号的增加。该检测方法的灵敏度达到4 pg菌丝DNA,整个检测过程控制在4 h内,其快速、特异和灵敏的特点可以满足进境油菜籽样品的快速初检以及病菌分离物的快速鉴定。  相似文献   

14.
Tests on samples of oilseed rape seed ( Brassica napus ) sown in the UK between 1981 and 1984 indicated that on average 25% of samples were infected with Alternaria brassicae and 61% with Leptosphaeria maculans , with maximum incidences of infection of 19 and 4.2% respectively. Much infection by Alternaria spp. occurred on vegetable and forage brassica seed produced in the UK between 1979 and 1983. In B. oleracea types A. brassicicola occurred most frequently, affecting 88% of samples and up to 55% of seeds. A. brassicae was detected in 44% of B. oleracea samples and in up to 13% of seeds. Little Alternaria infection occurred in swede or forage rape samples (B. napus ), but A. brassicae affected up to 8–5% of seeds in turnip samples ( B. campestris ). L. maculans occurred in 44% of samples of vegetable and forage brassica seed produced in the UK, with a maximum of 4–6% infected seeds. A. brassicicola was present in 73% of samples of imported B. oleracea seed, affecting up to 25.5% of seeds. A. brassicae was absent from these samples and little L. maculans was detected. Pathogenicity tests on isolates of L. maculans from infected seeds indicated that virulent pathotypes were present in 16 rape seed samples but in only one sample (swede) of vegetable or forage brassica seed. The high incidence of seed infection by these pathogens emphasizes the importance of applying fungicide treatments to all types of brassica seed.  相似文献   

15.
Quantitative resistance to Leptosphaeria maculans in Brassica napus was investigated in field and controlled environments using cultivars Darmor (with quantitative resistance) and Eurol (without quantitative resistance). In field experiments, numbers of phoma leaf spot lesions in autumn/winter and severity of stem canker the following summer were assessed in three growing seasons. There were no differences between Darmor and Eurol in number of leaf lesions in autumn/winter. However, stem cankers were less severe on Darmor than Eurol at harvest the following summer. In controlled-environment experiments, development of leaf lesions at different temperatures (5–25°C) and wetness durations (12–72 h) was investigated using ascospore inoculum; symptomless growth of L. maculans along leaf petioles towards the stem was quantified using quantitative PCR and visualized using GFP-expressing L. maculans ; growth of L. maculans within stem tissues was investigated using GFP-expressing L. maculans . There were more leaf lesions on Darmor than Eurol, although there was no difference between Darmor and Eurol in L. maculans incubation period. There were no differences between Darmor and Eurol in either distance grown by L. maculans along leaf petioles towards the stem or quantity of L. maculans DNA in leaf petioles, but L. maculans colonized stem tissues less extensively on Darmor than Eurol. It was concluded that quantitative resistance to L. maculans operates during colonization of B. napus stems by the pathogen.  相似文献   

16.
The survival of Leptosphaeria maculans , which causes phoma stem canker (blackleg), on oilseed rape residues ( Brassica napus ) in South Australia was investigated. Using a quantitative polymerase chain reaction (PCR) assay for L. maculans DNA, the pathogen was mainly detected in the upper 5 cm of the soil profile, including residues on the soil surface. As the size of organic matter particles in the soil decreased, so did the quantity of L. maculans detected in them. To obtain representative data for a field, at least 30 subsamples needed to be collected over the 0·81 ha area studied. In a survey of 49 commercial fields in South Australia, most L. maculans was detected in fields 1 year after oilseed rape had been grown, with less detected after 2 years and negligible amounts 3 years or more after cropping. The diagnostic DNA-based assay for L. maculans reduced the time and cost of studying L. maculans survival in soil and increased the sensitivity and accuracy of results compared with estimates of propagule number of colony-forming units on a semiselective medium.  相似文献   

17.
On oilseed rape, 207 leaf lesions attributed to Leptosphaeria maculans were classified as typical or atypical. Starch gel electrophoresis of glucose phosphate isomerase (GPI) performed on extracts of 229 leaf lesions comprising the 207 with L. maculans symptoms and 22 with Pseudocercosporella capsellae symptoms, yielded four different electrophoretic patterns of alloenzymes designated ET1 to ET4. In addition to ET1 and ET2, characteristic respectively of A- (highly virulent) and B- (weakly virulent) group isolates of L. maculans , the previously undescribed ET3 allozyme was recovered from a few typical and atypical L. maculans leaf lesions. The fastest ET4 allozyme was specific to P. capsellae . All but two typical leaf lesions produced the ET1 allozyme, whereas atypical lesions produced one of the three L. maculans allozymes. Occasionally a mixture of two allozymes was recovered from a same-leaf lesion. GPI electrophoresis performed directly on leaf lesions proved a useful and reliable method to identify L. maculans , and to differentiate between L. maculans and P. capsellae . This method of discrimination enabled deductions, from 377 leaf lesions analysed, about the structure of L. maculans populations on different oilseed rape varieties.  相似文献   

18.
Field experiments in Europe have shown that Chinese cultivars of winter oilseed rape ( Brassica napus ) are very susceptible to the pathogen Leptosphaeria maculans (cause of phoma stem canker). Climatic and agronomic conditions in China are suitable for L. maculans since the closely related but less damaging pathogen L. biglobosa occurs on the winter and spring oilseed rape crops there. Major gene resistance to L. maculans is not durable; when introduced into commercial oilseed rape cultivars it is rapidly rendered ineffective by changes in the pathogen population. The threat to Chinese oilseed rape production from L. maculans is illustrated by the way in which L. maculans has spread into other areas of the world where previously only L. biglobosa was present, such as Canada and Poland. Models were developed to describe the spread (in space and time) of L. maculans across Alberta province, Canada, based on survey data collected over a 15-year period. These models were used to estimate the potential spread of L. maculans across the Yangtze river oilseed rape growing areas of China and its associated costs. Short-term strategies to prevent occurrence of severe phoma stem canker epidemics in China include training of extension workers to recognise symptoms of the disease and use of PCR-based diagnostics to detect the pathogen on imported seed. Long-term strategies include the introduction of durable resistance to L. maculans into Chinese oilseed rape cultivars as a component of an integrated disease management programme. The costs of such strategies in relation to costs of a phoma stem canker epidemic are discussed.  相似文献   

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