小麦网腥黑穗病温室内人工接种方法的探索

小麦网腥黑穗病是由小麦网腥黑粉菌Tilletia caries引起,会导致小麦严重减产.为了获得小麦网腥黑穗病较高的发病率,本研究利用穗部接菌、土壤接菌、根部接菌3种方法进行温室内人工接种小麦.结果表明,利用穗部接菌及土壤接菌发病率分别为52.5％和10％,而根部接菌发病率仅有4％.利用穗部接菌可以获得更多的发病植株,...     

进境小麦中沙地牧草腥黑粉菌的鉴定

从上海口岸进境的澳大利亚小麦中发现一种类似小麦印度腥黑粉病菌的腥黑粉菌冬孢子，对该菌冬孢子进行了形态学特征和PCR检测，根据结果，将这种冬孢子鉴定为沙地牧草腥黑粉菌Tilletia ehghartaTle；本研究设计了T．ehrhartaea的特异引物Eh2/Eh4，结合引物Till／Til4建立了T．ehdmrtae的套式PCR检测方法。     

应用聚合酶链反应技术鉴定印度腥黑穗病菌

用一对专化于印度腥黑穗病菌的引物Ｔ１１７Ｍ１（５＇－ＴＣＣＣＣＴＴＧ－ＧＡＴＣＡＧＡＡＣＧＴＡ－３＇）和Ｔ１１７Ｍ２（５＇－ＡＧＡＡＧＴＣＴＡＡＣＴＣＣＣＣＣＣＴＣＴ－３＇）可特异地扩增印度腥黑穗病菌产生一段８２５ｂｐ的产物,而稻粒黑粉病菌则不能被扩增。实验还表明,用聚合酶链反应（ＰＣＲ）方法检测灵敏度可达到１００个未萌发的冬孢子,这为进口粮印度腥黑穗病菌的检疫提供了有力工具。     

异硫氰酸甲酯对小麦网腥黑穗病菌杀灭效果研究

在20℃和60%温湿度条件下,分别测定了氧硫化碳、异硫氰酸甲酯(Methyl isothiocyanate,MITC)等7种新型熏蒸剂对小麦网腥黑穗病菌(Tilletia caries(DC.)Tul.,TCT)的熏蒸效果。结果表明,除MITC外,其他6种熏蒸剂均不能完全杀灭TCT病菌;MITC熏蒸120h可有效杀灭TCT病菌,其LD90、LD95、LD99、LD99.9963分别为15.438、17.046、20.530、32.414g/m3。但是,由于小麦对MITC的高吸附性导致熏蒸剂空间浓度过低,使得有载物熏蒸试验中投药50、100g/m3MITC均未能有效杀灭小麦中的TCT病菌。残留检测结果表明,散气16d后小麦中MITC残留量分别降至0.29、1.07mg/kg,残留较高。结论:MITC对TCT有很好的杀灭效果,但由于小麦对MITC的吸附性很强,需进一步研究熏蒸方式,方可实际应用。     

套式PCR直接检测印度腥黑穗病菌冬孢子

用印度腥黑穗病菌冬孢子制备模板DNA ,利用印腥特异性引物T3 /T6,T3 /T4和套式PCR(nestPCR)扩增技术直接检测印腥冬孢子 ,检测的灵敏度可达 1个冬孢子。检测时间缩短为 1天。这种简单、快速、灵敏、实用和准确的PCR检测技术适用于口岸印腥检疫的需要 ,解决了常规PCR检测中DNA制备需要萌发冬孢子和检测时间长的难题。     

Real-time PCR法定量检测柑橘绿霉病菌对抑霉唑的抗性频率

 抑霉唑被广泛用来防治由指状青霉菌(Penicillium digitatum)引起的柑橘绿霉病。已有研究表明,柑橘绿霉病菌对抑霉唑的抗性由CYP51基因启动子区5个126-bp转录增强子的简单串联重复和126-bp转录增强子上199-bp的特异性片段插入所引起。基于这2种抗性分子机制,通过设计特异引物和优化条件,建立real-time PCR高通量分子检测技术,用于快速检测柑橘包装贮藏库中绿霉病菌群体对抑霉唑的抗性频率F_R,指导科学用药。     

水稻腥黑粉病菌的单孢检测

根据水稻腥黑粉菌两个聚类群与其它黑粉菌ITS序列的差异,分别设计了两个聚类群的特异引物Hor2/Hor9和Hm1/Hm5,结合ITS通用引物Til1/Til4建立了分别检测水稻腥黑粉菌两个聚类群单个冬孢子的套式(巢式)PCR检测方法,整个检测过程可以缩短至8h。     

小麦印度腥黑穗病菌PCR检测

应用PCR方法对小麦印度腥黑穗病菌及其近似种或相关种包括黑麦草腥黑粉菌、狼尾草腥黑粉菌、水稻腥黑粉菌等10种腥黑粉菌共14个菌株进行了检测研究。根据线粒体DNA的序列分别设计了扩增小麦印度腥黑穗病菌的特异性引物和扩增黑麦草腥黑粉菌的特异性引物，根据核糖体内转录区（ITS）DNA片段设计了扩增腥黑粉菌属真菌的引物，应用PCR方法能将小麦印度腥黑穗病菌与黑麦草腥黑粉菌及其它近似种或相关种加以区分。本方法稳定、可靠、重复性强，已分别在不同实验室的不同型号PCR仪上得到验证。     

应用real-time PCR定量检测小麦条锈菌潜伏侵染量方法的建立

 小麦条锈病是我国小麦主要病害之一。快速、及时地诊断与定量监测处于潜育状态下的病叶,对准确估计越冬、越夏后的病情,制定正确的防治方案具有重要的意义。根据小麦条锈菌Puccinia striiformis的β-tubilin基因序列设计对该病原菌的种具有特异性的引物betaf/betar,并分别在普通PCR和real-time PCR扩增时对该引物的特异性和灵敏性进行了测定。结果表明该引物对小麦条锈菌特异性高,可稳定扩增出243 bp的目标条带。Real-time PCR的灵敏度为普通PCR的100倍。应用此特异性引物,建立了real-time PCR测定系统,定量测定了条锈菌在小麦叶片接种后组织内的DNA随时间的变化。结果表明,在接种后12 h,可在小麦叶片内检测到条锈菌,且条锈菌在小麦叶片内潜育期间随时间呈指数增长。接种第6 d后叶片内的菌量有明显的增加。建立的小麦条锈菌的real-time PCR早期定量测定方法,为及时、快速监测小麦条锈病在潜育期间的发病规律以及为该病的预测、防治提供依据。     

温度对小麦矮腥黑穗病菌冬孢子萌发的影响

温度对小麦矮腥黑穗病菌冬孢子萌发影响的试验结果表明,TCK冬孢子在-2～12℃范围内都可以萌发,5℃为最佳萌发温度。同一分离菌冬孢子在不同温度处理的萌发特性和萌发率有差异;不同分离菌冬孢子在相同温度下的萌发特性和萌发率有很大差异。在5℃下5个分离菌中,冬孢子萌发起始时间最短的为15 d,最长为35 d,培养60 d时分离菌T_t1、T_t2、T_y、T_m和T_u冬孢子的萌发率分别为86.4%、23.9%、23.3%、44.3%和81.0%。根据分离菌T_t2不同温度下培养50 d时的萌发率,建立了萌发率(Y)与温度(X)的模型为Y=0.150 8 exp[-0.02949(X-4.957 6)]2,此结果为TCK的风险评估提供了基础数据。     

土壤湿度对小麦矮腥黑穗病菌(TCK)冬孢子萌发的影响

通过土壤湿度对小麦矮腥黑穗病菌(Tilletiacontronversa Kühn,TCK)萌发率的影响试验研究表明,其冬孢子在土壤质量含水量为1%～28%(相对含水量3.57%～100%)范围内均可萌发,其适宜萌发的土壤质量含水量范围为10%～25%(相对含水量17.85%～89.3%),最佳土壤相对含水量范围在65%～75%之间;不同分离物在相同土壤湿度培养下,多数分离物冬孢子的萌发率之间差异不显著。根据分离菌T_t1和T_t2在不同土壤湿度下培养50d和60d的冬孢子萌发率,建立了TCK冬孢子萌发率与土壤相对含水量的关系模型,此结果为TCK的风险评估提供了基础数据。     

A multiplex real-time PCR assay enables simultaneous rapid detection and quantification of bacteria associated with acute oak decline

Acute oak decline (AOD) is a syndrome affecting mature oak trees and is characterized by stem bleeds from vertical fissures on trunks, and inner bark necrosis caused by a polybacterial consortium, in which Gibbsiella quercinecans and Brenneria goodwinii, and to a lesser extent Rahnella victoriana and Lonsdalea britannica, play key roles. Here we report a novel multiplex real-time PCR assay that enables simultaneous and rapid detection and quantification of these four bacterial species from stem bleed swabs. Experiments with axenic cultures were performed to determine specificity and sensitivity of the multiplex quantitative PCR (qPCR). Whilst the primer/probe set for B. goodwinii was species-specific, primer/probe sets for the other three species were able to identify other members of their respective genera. There was no cross detection of genera within the multiplex qPCR, and non-target bacteria were not detected. The multiplex AOD assay had differential sensitivity for each bacterial species. The assay was evaluated on swab samples collected from stem bleeds of declining oak trees at a site in south-east England and was able to detect all four bacterial species. Absolute quantification of the bacteria from swab samples was possible through the inclusion of a standard curve prepared from dilutions of gene copy standards. This diagnostic tool will facilitate rapid detection of AOD-associated bacteria from samples that can easily be taken by non-specialists without specific training, and will also find application in other experimental work such as pathogenicity and control trials.     

Real-time quantitative PCR assays for quantification of L1781 ACCase inhibitor resistance allele in leaf and seed pools of Lolium populations

The I1781L amino acid substitution in the target ACCase enzyme causes broad resistance to ACCase inhibitor herbicides in several monocotyledenous weeds of agronomic importance. This mutation results from a substitution of an adenine (A) residue by either a thymine (T) or cytosine (C) at position 5341 in Alopecurus myosuroides Huds and at an equivalent position in Lolium species, Avena fatua L. and Setaria viridis (L.) Beauv. Two different procedures, the PCR-based allele-specific assay (ASA) and the derived cleaved amplified polymorphic sequence (dCAPS) method, have previously been described for detecting this mutation. These methods are, however, only amenable to low sample throughput and are used in the analysis of single plants. Here, an alternative high-throughput ARMS/Scorpion real-time quantitative PCR (Q-PCR) method for measuring levels of the I1781L mutation in pools of leaf and seed samples of Lolium populations is presented. The limit of detection for C and T mutant alleles in a background of wild-type A is 0.02 and 0.0003% respectively. In this study, DNA from batches of 24 leaf segments measuring 0.5 cm from different plants or 1000 seeds could be conveniently extracted and accurately analysed. As part of assay validation, the comparative analysis of five geographically distinct Lolium populations with dCAPS and Q-PCR procedures demonstrated the accuracy of the latter method, and the three possible II1781, IL1781 and LL1781 ACCase genotypes being distributed as predicted by the Hardy-Weinberg principle. Given the dominance of the L1781 over the I1781 allele at recommended field rates for most ACCase inhibitors, the frequency of herbicide survivors in the field owing only to the presence of the I1781L mutation is thus predicted to be 2pq + q(2), where p and q are the frequencies of the I1781 and L1781 alleles as determined by Q-PCR. The Q-PCR assay established allows detection of very low levels of the L1781 ACCase mutation before resistance would normally be discernible in the field. Therefore, it offers the opportunity to tackle resistance at its very onset, potentially avoiding implementation of complicated and often costly weed management practices.     

小麦矮化腥黑穗病菌冬孢子的定量检测及几种计数方法的比较

用人工污染有小麦矮化腥黑穗病菌Tilletia controversa Kuehn冬孢子的小麦样品，按国家标准GB／T18085－2000规定的洗涤程序，对TCK的定量检测以及3种冬孢子计数方法进行了比较研究，并建立了定量评估样品中孢子量的方法。研究表明，使用国际种子检验协会（ISTA）的标准种子洗涤方法仅能发现30．72％－46．58％的TCK孢子，检验洗涤后的小麦表面，仍可分析有大量孢子沾附于小麦种子表面。应用回归分析，建立了定量评估样品中孢子的方程Y＝2．0131（X＋2）^1.0325-2。通过比较3种孢子计数方法，认为依据国标建立的计数方法得到的结果变异最小。     

Development of a PCR-based diagnostic tool specific to wheat dwarf bunt,caused by <Emphasis Type="Italic">Tilletia controversa</Emphasis>

Wheat dwarf bunt, one of the important international quarantine diseases, is caused by Tilletia controversa. Tilletia caries is a close relative species of T. controversa and the teliospore morphology and genomic structure of T. caries are very similar to those of T. controversa. In order to distinguish between them, a random amplified polymorphic DNA (RAPD) primer-mediated asymmetric-PCR (RM-PCR) was developed to screen differential sequences between the two pathogens. By RM-PCR, a 1,322 bp DNA fragment (PR32) was selected from 18 T. controversa and 29 T. caries strains. The PR32 genes were specific for T. controversa and almost had no homology to T. caries or other fungi in the present database. With primers designed from PR32, all 18 T. controversa strains were amplified, but no bands appeared in 29 T. caries strains by classical PCR. To identify T. controversa rapidly and accurately, SYBR Green I and TaqMan probe real-time PCR were established based on PR32. With TaqMan Real-Time PCR, different T. controversa strains and T. caries strains were detected. The results showed that all T. controversa strains were amplified with Ct from 19–29 and amplified curves were obtained. In contrast, the amplification of all T. caries strains did not show any signals, without Ct and amplified curves. Moreover, the developed TaqMan real-time PCR was used to detect T. controversa from asymptomatic wheat tissues successfully.     

葡萄根瘤蚜TaqMan-MGB实时荧光定量检测方法的研究

为建立葡萄根瘤蚜实时荧光定量PCR的检测方法,参考Karen Herbert等设计的特异性引物与TaqMan-MGB荧光探针,构建以标准阳性质粒作为标准品制作标准曲线,并经优化反应条件,建立葡萄根瘤蚜的实时荧光PCR绝对定量检测方法,进行敏感性和重复性试验,并对受葡萄根瘤蚜为害的葡萄根际土壤进行初步定性检测.结果表明:该方法的灵敏度可达1.625拷贝/μL,3次重复检测的变异系数均小于5％.提取0.25g含有10头葡萄根瘤蚜若虫土壤的DNA,并将其梯度稀释,用建立的荧光定量PCR进行检测,将DNA稀释103倍后,仍能检测出阳性结果.对受害葡萄根际土壤检测结果为阳性.葡萄根瘤蚜TaqMan-MGB探针实时荧光PCR检测技术具有特异性强,敏感性高,易操作等优点,有很好的应用前景和研究价值.     

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?     

A real-time PCR assay for detection and quantification of Verticillium dahliae in spinach seed

Verticillium dahliae is a soilborne fungus that causes Verticillium wilt on multiple crops in central coastal California. Although spinach crops grown in this region for fresh and processing commercial production do not display Verticillium wilt symptoms, spinach seeds produced in the United States or Europe are commonly infected with V. dahliae. Planting of the infected seed increases the soil inoculum density and may introduce exotic strains that contribute to Verticillium wilt epidemics on lettuce and other crops grown in rotation with spinach. A sensitive, rapid, and reliable method for quantification of V. dahliae in spinach seed may help identify highly infected lots, curtail their planting, and minimize the spread of exotic strains via spinach seed. In this study, a quantitative real-time polymerase chain reaction (qPCR) assay was optimized and employed for detection and quantification of V. dahliae in spinach germplasm and 15 commercial spinach seed lots. The assay used a previously reported V. dahliae-specific primer pair (VertBt-F and VertBt-R) and an analytical mill for grinding tough spinach seed for DNA extraction. The assay enabled reliable quantification of V. dahliae in spinach seed, with a sensitivity limit of ≈1 infected seed per 100 (1.3% infection in a seed lot). The quantification was highly reproducible between replicate samples of a seed lot and in different real-time PCR instruments. When tested on commercial seed lots, a pathogen DNA content corresponding to a quantification cycle value of ≥31 corresponded with a percent seed infection of ≤1.3%. The assay is useful in qualitatively assessing seed lots for V. dahliae infection levels, and the results of the assay can be helpful to guide decisions on whether to apply seed treatments.     

Rapid-cycle PCR detection of Pyrenophora graminea from barley seed

Sixty RAPD primers were used to screen for a diagnostic marker that could be used to identify Pyrenophora graminea , a fungal seedborne pathogen that causes leaf stripe on barley. Primer pairs were designed to differentiate P. graminea from other Pyrenophora spp. using a sequence-characterized amplified region (SCAR) approach. A pair of P. graminea -specific primers (PG2 F/R) was obtained that amplified a single fragment from 37 isolates of P. graminea tested, but not from 29 isolates of other Pyrenophora spp. or 12 saprophytes isolated from barley seed. Rapid PCR detection was achieved using a LightCycler, in which the emission of fluorescence from the binding of SYBR Green I dye to the PCR products is measured. The P. graminea -specific product resulting from amplification with PG2 F/R can be distinguished from any nonspecific products by post-PCR melting point analysis. The PCR assay involves 40 amplification cycles of PCR, and the total PCR test including melting point analysis takes 25 min to complete. The rapidity of this test, combined with the closed 'in-tube' detection of PCR products, which reduces the potential for contamination, offers significant advantages compared with conventional laboratory and PCR analyses.     

基于TaqMan MGB探针的黑白轮枝菌检测方法

根据黑白轮枝菌(Verticillium albo-atrum)及其近似种β-微管蛋白基因(β-tubulin)序列差异,设计并合成1对引物和1条Taq Man-MGB探针,建立了黑白轮枝菌的实时荧光PCR检测方法。对供试黑白轮枝菌及其近似种实验表明,该方法特异性强,只有黑白轮枝菌可被检出。通过对反应体系的优化,确定了最佳反应条件:引物终浓度为1.0μmol/L,探针终浓度为0.7μmol/L。灵敏度试验结果显示,最低检测限量为总DNA含量10 pg(20μL反应体系)。此方法快速灵敏,为快速检测黑白轮枝菌提供了重要参考。