进境油菜籽中黑胫病菌和茎基溃疡病菌的检测

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。     

油菜茎基溃疡病菌的实时荧光PCR检测

 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。     

油菜茎基溃疡病菌在中国定殖的可能性评估

为明确油菜茎基溃疡病菌Leptosphaeria maculans在我国定殖的可能性,并制订针对性的检疫措施,本研究基于该病菌在英国、法国、德国、波兰、加拿大、澳大利亚的分布数据,选取与病害发生有关的温度、降水量等15个变量,利用Max Ent和GARP两种生态位模型预测其在其它地区的潜在分布,并利用受试者工作特征曲线(ROC)来评价模型模拟精度。结果显示,2种模型均能够较好地预测L.maculans的分布区域,与已记载的分布区域高度吻合,且全球范围内存在着许多高度适合L.maculans定殖的地区;2种模型也能预测L.maculans在我国的潜在分布区域,并且预测结果一致;GARP结果显示L.maculans在我国的中高度适生区包括内蒙古、吉林、陕西、宁夏、甘肃、新疆、西藏等地。鉴于L.maculans在我国的潜在分布研究结果,建议完善疫情监测体系,采取措施控制病菌的"进入风险",降低病菌"进入"后定殖的可能性。     

油菜茎基溃疡病菌LAMP-LFD检测方法的建立

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。     

壳聚糖对水稻恶苗病菌和油菜菌核病菌的作用

用平板含毒介质法测定了壳聚糖对水稻恶苗病菌(Fusarium moniliforme)和油菜菌核病菌(Sclerotinia sclerotiorum)的影响。结果表明,壳聚糖对F.moniliforme抑制作用相对较强,EC_(50)低于1000mg/L,而对S.sclerotiorum的作用较弱,EC_(50)高于3000mg/L。壳聚糖抑制F.moniliforme菌丝生长,导致菌丝畸形肿胀,产生不正常分支,并且抑制其分生孢子的萌发和芽管伸长。壳聚糖处理后F.moniliforme菌丝细胞膜的透性增加,导致蛋白质渗漏,但对其细胞膜的主要结构成分麦角甾醇的含量影响不明显。在离体培养时,壳聚糖对S.sclerotiorum产草酸毒素无明显影响,但预先经壳聚糖处理后,再接种S.sclerotiorum的油菜叶片中草酸含量显著减少。     

进境澳大利亚大麦夹杂油菜茎基溃疡病菌的检疫鉴定

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?     

油菜菌核病菌对多菌灵和乙霉威的抗药性机理

生物测定结果表明,油菜菌核病菌田间菌株对多菌灵(MBC)敏感性表型呈多样性,即存在MBCS、MBCLRLR、MBCHRHR和MBCVHR表型.而对乙霉威(DIE)则只检测到DIES和DIEHR表型.MBCS、MBCLR和MBCHR菌株中除JD2-3菌株为DIES外,其余菌株均为DIEHR,MBCVHR菌株对DIE表现为DIES.MBC和DIE之间存在典型的负相关交互抗性.序列分析结果表明,表型为MBCVHRDIES菌株的β-微管蛋白基因,第198位氨基酸由Glu(GAG)突变为Ala(GCG);表型为MBCHRDIEHR菌株的β-微管蛋白基因的突变位点在第200位,由Phe(TTC)突变为Tyr(TAC);而表型为MBCSDIES和MBCLRDIEHR的菌株在所扩增的β-微管蛋白基因片段中未发生突变.初步表明,β-微管蛋白基因198和200位氨基酸的突变是引起油菜菌核病菌对多菌灵抗药性呈多样性的分子机理.     

进口油菜籽中十字花科小球腔菌检测方法概述

十字花科小球腔菌(Leptosphaeria maculans)是影响油菜生产的重要病原真菌,主要分布在加拿大、澳大利亚、美国等油菜主产区.我国尚无此病原危害的报道.该病原通过带菌种子作远距离传播传入新发病区,加强该病原菌的检测是防范该有害生物传入的重要手段.本文对该病原菌的现有检测鉴定方法进行了综述,为口岸部门开展该病原菌的检测以及制定相应检测方法标准提供借鉴.     

土壤中添加蚯蚓及秸秆对油菜积累镉的影响

采用盆栽试验,在不同镉(Cd)污染浓度的灰漠土中分别添加蚯蚓和新鲜秸秆,研究了添加蚯蚓和秸秆还田条件下油菜(Brassica rapa L.spp.Chinenesis)-土壤体系中Cd的分布情况.结果发现:(1)添蚯蚓可有效增加土壤中Cd向油菜体内转移;蚯蚓可以在Cd污染的土壤中生存,蚯蚓体内Cd浓度随土壤环境中Cd...     

盾壳霉控制油菜菌核病菌再侵染及其叶面存活动态的研究

 本文评估了施于油菜(Brassica napus)叶片上的盾壳霉(Coniothyrium minitans)控制油菜菌核病菌再侵染能力,探讨了其作用机理,并测定了盾壳霉分生孢子在油菜叶面上的存活动态。结果如下:叶面上的盾壳霉对油菜菌核病菌的初侵染影响较小,但在高剂量(> 10⁶孢子/ml)时可以控制病斑的扩展。所有供试剂量的盾壳霉均可不同程度地控制再侵染。盾壳霉分生孢子可在叶面病部迅速萌发,48 h和72 h时孢子萌发率分别为51%和95%,而在健康叶面上6 d未能检测到萌发的孢子。自携带盾壳霉的叶面病部不能分离到核盘菌,表明叶面上的盾壳霉已寄生并破坏了核盘菌再侵染菌丝。自油菜叶面上分离到的盾壳霉菌落数随时间延长而降低,但其分生孢子至少可以在叶面上存活28 d。这即表明,在叶面上适时适量地添加盾壳霉可以控制油菜菌核病的为害。     

进境澳大利亚油菜籽中茎基溃疡病菌的检测

 41 fungal isolates with similar morphological characteristics to Leptosphaeria maculans were obtained by the deep-freezing filter paper method from 2100 seeds of Brassica napus imported from Australia.The isolate 8129-5 showed a slower growth on PDA at 20℃with growth rate of 2.8 mm/day.The colonies on PDA at 20℃ had an irregular or regular margin with white or grayish white compact aerial mycelium.No diffusible pigment was produced on PDA at 31℃ or in liquid Czapek-Dox media at 20℃.PCR detection showed that the isolate 8129-5 could be amplified by L.maculans-specific primers LmacF/LmacR and got expected product of 331 bp.The sequence analysis revealed that the ITS sequence of isolate 8129-5 had 99.8% identity with L.maculans.Pathogenicity of the isolate 8129-5 was confirmed on cotyledons of rape seed by artificial inoculation compared with typical symptom of L.maculans.Based on the morphological characteristics, PCR detection and the result of pathogenicity test, the isolate 8129-5 was identified as L.maculans.     

Epidemiology and management of Leptosphaeria maculans (phoma stem canker) on oilseed rape in Australia, Canada and Europe

Phoma stem canker (blackleg), caused by Leptosphaeria maculans , is an important disease on oilseed rape (canola, rapeseed, Brassica napus , Brassica juncea , Brassica rapa ) causing seedling death, lodging or early senescence in Australia, Canada and Europe, but not in China. The two forms of L. maculans (A group and B group) that occur on oilseed rape are now considered to be separate species. The epidemiology and severity of phoma stem canker differs between continents due to differences in the pathogen population structure, oilseed rape species and cultivars grown, climate and agricultural practices. Epidemics are most severe in Australia, where only the A group occurs, and can be damaging in Canada and western Europe, where both A and B groups occur, although their proportions vary within regions and throughout the year. Epidemics are slight in China, where the A group has not been found. Dry climates (Australia, western Canada) lengthen the persistence of infected debris and may synchronize the release of airborne ascospores (after rain) with seedling emergence. L. maculans spreads from cotyledon and leaf infections down petioles to reach the stem, with infections on cotyledons and leaves early in the season producing the most damaging stem cankers at the stem base (crown). Development of both crown cankers and phoma stem lesions higher up stems is most rapid in regions with high temperatures from flowering to harvest, such as Australia and Canada. Breeding for resistance (genetic, disease escape or tolerance), stubble management, crop rotation and fungicide seed treatments are important strategies for control of phoma stem canker in all areas. Fungicide spray treatments are justified only in regions such as western Europe where high yields are obtained, and accurate forecasts of epidemic severity are needed to optimize their use.     

环介导等温扩增技术检测大丽轮枝菌

 本研究基于环介导等温扩增技术(loop-mediated isothermal amplification,LAMP),通过比对分析大丽轮枝菌(Verticillium dahliae)与其相近种不同靶标序列间的差异,选取Gpd(glyceraldehyde-3-phosphate dehydrogenase,甘油醛-3-磷酸脱氢酶)基因作为靶标基因,设计并筛选了四条特异性强、灵敏度高的LAMP引物和两条环引物,建立了一种基于颜色判定的简单、快速和灵敏的大丽轮枝菌的检测方法,并进行了特异性、灵敏度实验及田间发病组织的检测。该方法在62 ℃等温条件下进行核酸扩增反应70 min,扩增前加入染料HNB(羟基萘酚蓝),反应后根据染料颜色变化判定扩增结果。特异性试验中,仅含有大丽轮枝菌菌株DNA的反应管扩增后呈天蓝色的阳性反应,而其他供试菌株均呈紫色的阴性反应。该方法的最低检测限为100 pg·μL^-1,在土壤中检测的灵敏度为10个孢子/0.25g土壤。该技术能够检测出棉花发病组织中的目标菌,对采自江苏和山东的24份疑似病害样本进行检测,11份为阳性。该方法的建立为大丽轮枝菌的检测及其所致病害的诊断提供了新的技术。     

Differentiating A and B groups of Leptosphaeria maculans, causal agent of stem canker (blackleg) of oilseed rape

Stem canker or blackleg of brassicas, caused by Leptosphaeria maculans , is one of the most damaging diseases of winter oilseed rape in the UK. Airborne ascospores, released in autumn and winter, initiate leaf infections which may lead to colonization of the petiole and, later in the season, formation of stem lesions and cankers. Although isolates of the pathogen differ in ability to cause damaging stem cankers, this is not readily apparent from leaf spotting or stem lesion symptoms. However, several cultural, biochemical and genetic characteristics appear to be associated with the ability to form damaging stem cankers and isolates can be assigned to one of two groups, termed A and B, on the basis of differences in these characteristics. To investigate the relationship between leaf spotting symptoms and subsequent stem canker formation, and to improve understanding of the epidemiology of this pathogen, it is desirable to differentiate between the stem canker forming A group and the less damaging B group of L. maculans . Characterization of isolate type is also important in seed testing and crop breeding programs, particularly in countries such as Canada and Poland where the A type is not ubiquitous. This article reviews methods, including plant assays, assessments of growth characteristics in vitro , isozyme analyses, secondary metabolite profiling, serology, and nucleic acid analyses, that can be used to differentiate the A and B groups.     

Splash dispersal of Leptosphaeria maculans pycnidiospores and the spread of blackleg on oilseed rape

The fungus Leptosphaeria maculans causes blackleg (phoma stem canker), one of the most serious diseases of oilseed rape. The role of pycnidiospores produced during asexual reproduction is poorly documented and limits the understanding of the pathogen's population dynamics. The objectives of this study were to assess rain-splash dispersal of pycnidiospores of L. maculans from phoma leaf spots, and transmission of the disease from oilseed rape stubble carrying pycnidia. The work was conducted in still air with either a drop generator or a rain simulator. The impact of simulated incident drops on phoma leaf spots resulted in the dispersal of L. maculans pycnidiospores within splash droplets. Ninety per cent of the spores were collected within 14 cm of the source and a few were regularly observed up to 40 cm. Pycnidiospores produced on oilseed rape stubble and dispersed by simulated rain infected oilseed rape trap plants in a spatial pattern that matched the spatial dispersal of the pycnidiospores. In the field, rain-splash dispersal of pycnidiospores could increase the pathogen population and may enhance sexual reproduction by facilitating the mating of initially spatially separated isolates of opposite mating type.     

Strategies to prevent spread of Leptosphaeria maculans (phoma stem canker) onto oilseed rape crops in China; costs and benefits

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.     

Survival of Leptosphaeria maculans in soil on residues of Brassica napus in South Australia

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.     

快速检测马铃薯干腐病病原接骨木镰孢的环介导等温扩增技术

 本研究基于环介导等温扩增技术(loop-mediated isothermal amplification, LAMP),建立了一种快速、准确和灵敏的接骨木镰孢的检测技术。通过比对接骨木镰孢与其近源种之间的候选靶标序列,选取TEF1-α(translation elongation factor 1-α,翻译延伸因子)基因作为靶标,设计并筛选出一套对该病原菌具有种特异性的LAMP引物,建立了检测接骨木镰孢的 LAMP 体系。该体系在反应前加入染料羟基萘酚蓝(hydroxynaphthol blue,HNB),经62℃恒温反应70 min之后,可根据肉眼观察到的反应物的颜色判定结果。特异性分析结果表明,仅接骨木镰孢的DNA经检测后呈天蓝色的阳性反应,而其他供试菌株的DNA均呈紫色的阴性反应。该方法对DNA的最低检测限为100 pg·μL^-1。在采自内蒙古和黑龙江的28份马铃薯干腐病疑似病害样本中,检测到14份阳性样品。该方法的建立为接骨木镰孢的检测及其所致病害的诊断提供了快捷准确的技术。