真菌病害是制约水稻高产稳产的重要障碍因素之一.为了研究水稻种子中真菌发生情况,对分离自水稻种子中的一株真菌进行系统发育分析及功能评价.在形态学鉴定基础上,以通用引物ITS1/ITS4对目标真菌序列进行PCR扩增、序列对比分析、邻接法(NJ)构建系统发育树.通过菌落培养特征、孢子形态特征观察,分离菌株S-51的形态特征与文献中雪霉叶枯病菌(Microdochium nivale)的描述极为相似;序列比对结果表明,目标真菌与雪霉叶枯病菌(M.nivale)相近,相似率为100％;系统发育树显示,其与雪霉叶枯病菌株在同一条分支上,雪霉叶枯病菌M.nivale和M.musae的亲缘关系最近.利用ITS片段开展的序列分析和系统进化树构建,结合形态特征,能够确定分离自水稻种子中的S-51菌株为雪霉叶枯病菌(M.nivale).

对26份水稻材料进行抗病基因检测,包括8个白叶枯病抗性基因(Xa1、xa5、Xa7、xa13、Xa21、Xa23、Xa26、Xa27)和2个稻飞虱病抗性基因(Bph14、Bph15).其中23份水稻材料中含有Xa1基因材料20份,含xa5基因材料20份,含Xa7基因材料23份,含xa13基因材料12份,含Xa21基因材料9份,含Xa23基因材料23份,其中抗性4份,感性19份,含Xa26基因材料23份,含Xa27基因材料21份,其中抗性5份,感性16份.另有3份材料进行稻飞虱病抗性基因Bph14、Bph15检测,检测结果表明这3份材料均含有Bph14、Bph15基因.选取13个已报道的基因标记或连锁标记检测26份杂交稻材料,初步确定了其抗白叶枯病和抗稻飞虱病等位基因分布和利用情况,为水稻基础材料的筛选、亲本材料的选择及育种应用提供了良好的参考.

2020年12月17日,9台二手小汽车登上"格罗勇气"轮发往埃及,这是连云港港首单二手小汽车出口业务.在国家二手车出口政策出台以后,从2019年下半年以来,连云港港积极开拓二手车出口市场,出口的二手车基本都是从山东等二手车市场经过整备后的二手重卡,主要装载到件杂货船上发往非洲市场,高峰时间每月出口400台左右.

To improve the quantitative detection efficiency of transgenic rice,and promote the implementation of transgenic food quantitative identification system,the components of transgenic rice ‘ Bt shanyou 63 ’ were analyzed with droplet digital PCR quantitative detection method.Based on the exogenous inserting sequence of transgenic rice ‘Bt shanyou 63’ and the rice sucrose phosphate synthase (SPS) gene,high-efficiency primer probes were selected,designed and synthesized to amplify the endogenous gene and the strain specific sequence of ‘Bt shanyou 63’ with droplet digital PCR.The result of primer probes optimizing experiment demonstrated that SPS60-F/R/P and ‘Bt shanyou 63’-F/R/P was suitable for quantitatively detecting the components of ‘Bt shanyou 63’ with dual channel ddPCR method.The accuracy verification experiment showed that the measured value was similar to the standard value,and the biases were 0.03,0.15,0.15 and 0.03,respectively.SD of measured value was 0.08-0.48,RSD was 4.86％-15.10％,and they were less than the requirement of European Union,indicating that the accuracy was good.The established detection system of droplet ddPCR method for transgenic rice line ‘Bt 63’ quantification is simple,high efficient and accurate,and it can be used for quantitative analysis of transgenic rice line ‘ Bt 63’ in agricultural products and foods.

本实验用SSR分子标记技术鉴定两系杂交水稻“C两优513”和“深两优5814”的种子纯度.本研究从48对SSR引物中筛选到3组引物(RM277、RM17、RM7389),选取“C两优513”和“深两优5814”种子各5组,每组样品随机取192粒进行纯度检测.针对同一份样品,SSR标记与田间鉴定结果均接近,相差最大一组差异为3.3％,最小差异0.04％.此外,不同引物对同一样品的鉴定结果通常存在差异.结果表明,SSR标记技术适用于“C两优513”和“深两优5814”的种子纯度鉴定.为控制检测误差,宜用多对引物同时进行实验.

Ustilaginoidea virens is an important fungus that causes rice false smut disease. This disease significantly reduces both grain yield and quality. Various methods have been developed for the detection of U. virens but most of these methods need sophisticated equipment such as a thermal cycler. Here, we present a loop-mediated isothermal amplification (LAMP) assay for the specific detection of U. virens. This assay used a specific region of the UvG-β1 gene (212-bp region) to design six LAMP primers. The LAMP assay was optimized by the combination of rapidity, simplicity, and high sensitivity for the detection of about 1 pg of target genomic DNA in the reaction whereas, with polymerase chain reaction (PCR), there was no amplification of DNA with concentrations less than 1 ng. Among the genomic DNA of 22 fungus species and two strains of U. virens, only the tube containing the DNA of U. virens changed to yellowish green with SYBR Green I. The color change was indicative of DNA amplification. No DNA was amplified from either the other 22 fungus species or the negative control. Moreover, 20 spikelets and 22 rice seed samples were used for the detection of rice false smut via LAMP. The results were comparable with conventional PCR. We conclude that gene UvG-β1 coupled with LAMP assay, can be used for the detection and identification of U. virens gene via LAMP.

Callosobruchus maculatus are frequently intercepted in quarantine at port.In this paper,516 bp base sequence of COI gene of C.maculatus and allied species was detected and analyzed.The results showed that there are 353 conseved sites,163 mutation sites,134 parsimony information sites and 29 singletons sites in these genes.Model Kimura 2-parameter was used to analyse the genetic distance,and the results revealed that the genetic distance among the same species varied from 0.001 to 0.013 with the average value of 0.008,while that among different species ranged from 0.114 to 0.193 with the average value of 0.161.Neighbor-joining phylogenetic tree was built based on the COI gene sequences,and the result showed that the same species was gathered in the same branch,with the bootstrap value of 100％.The results indicated that the molecular identification of C.maculatus and its allied species is practicable based on COI gene sequence.

出口工业品质量安全示范区是新公共服务理论的有效实践,为集群产业升级发展提供了路径支撑,是产业生命周期理论的延续.从理论联系实际角度,通过对天长示范区公共服务体系的实证分析,认为示范区公共服务体系改善了信息不对称、多元治理能力不足、跨界协作偏弱的先天不足,指出该示范区存在财政投入资金、产业结构、企业家精神、资源整合四方面不足,并给出了产业联动、政府职能转变、厚植创业文化、发展共享经济等建议,为下一步如何发展好示范区公共服务体系打下了基础.

采用基质辅助激光解析电离飞行时间质谱(matrix-assisted laser desorption/ionization time of flight mass spectrometry,简称MALDI-TOF-MS)技术对水稻作物的3种主要病原真菌进行分析,以获得稳定的指纹图谱.从菌物预处理方法、基质、点样方法等3个方面进行比较,对影响MALDI-TOF-MS分析结果的主要因素进行优化.结果表明,菌物预处理方法对检测结果的影响最大,热处理法在细胞壁较厚的真菌样品处理中可以获得较完整的生物信息;构建稻粒黑粉病病菌(Tilletia horrida)、稻曲病病菌(Ustilaginoidea virens)、恶苗病病菌(Fusarium moniliforme)的MALDI-TOF-MS鉴定规范化方法,扩充MALDI-TOF-MS指纹图谱数据库,可简便、快速、准确地对细胞壁加厚的真菌样品进行鉴定.

目的:建立Illumina Miseq深度测序筛查酸奶中微生物的方法,分析酸奶中微生物的多样性.方法:以9种酸奶样品为研究对象,提取酸奶中细菌基因组DNA,应用高通量测序技术测定细菌的16S rDNA v1-v3变异区序列,得到9种样品中微生物群体分布和丰度、以及不同样品间的菌种差异与进化关系.结果:酸奶中微生物主要为厚壁菌门(Firmicutes),占据99.6％之高,其中厚壁菌门主要以链球菌属(Streptococcus、87.1％)、乳杆菌属(Lactobacillus、10.3％)、乳球菌属(Lactococcus、0.3％)组成,9种酸奶中有3种同时含有链球菌属和乳杆菌属,其余6种样品中链球菌属几乎占据全部(＞97％).结论:Illumina Miseq深度测序技术可快速精确深入掌握酸奶中微生物多样性,结果对比显示不同酸奶菌种同质化程度高,其中链球菌属占绝对优势,同时发现部分酸奶标签标示不符.

A simple and rapid method for the detection of Tilletia horrida , the causal agent of rice kernel smut, in rice seeds is developed based on specific polymerase chain reaction (PCR). To design the specific primers for the detection of T. horrida , partial sequences of internal transcribed spacer (ITS) DNA region of T. horrida , T. controversa , T. walkeri , T. ehrhartae , T. indica and T. caries were analyzed and compared. A 503-bp fragment was amplified with the designed primers from the T. horrida genomic DNA. However, no PCR product was obtained from the DNA of other five Tilletia species and 22 fungal plant pathogens tested in the present work indicating the specificity of the primers for the detection of T. horrida . The PCR was performed by directly using the spores, isolated from the 21 different rice seed samples, as template DNA. The T. horrida was detected in 6 of the samples, indicating that 28.6% of the rice samples were contaminated with the kernel smut pathogen. This simple PCR based diagnostic assay can be applied for the direct and rapid detection and identification of T. horrida to screen large numbers of rice seed samples.

[目的]研究蜂蜜中蛋白质提取的有效方法,优化蜂蜜蛋白电泳条件.[方法]该试验使用丙酮、钨酸钠、硫酸铵、尿素-硫脲4种方法提取蜂蜜中的蛋白并进行比较,然后进行双向电泳分析,对影响双向蛋白电泳的IPG胶条的选择、蛋白质上样量、等电聚焦时间等主要因素进行优化.[结果]试验表明,采用硫脲法提取蜂蜜蛋白获得的蛋白含量最高,硫脲法和丙酮沉淀法获得的蛋白纯度最好,而硫脲法因干扰物质较少、易于裂解适于双向电泳技术,采用硫脲法提取的蜂蜜蛋白选择IPG胶条pH 5～8,蛋白上样量为150μg,等电聚焦时间达到32000 V·h的情况下能充分地聚焦,获得的双向电泳图谱最佳,优化后的洋槐蜜双向电泳图谱上可检测到326个蛋白点,重复性和分辨率均较好.[结论]蜂蜜类含糖样品中使用硫脲法提取蛋白能减少糖类物质干扰.

黑斑病是砀山梨上最重要的病害之一,建立一套砀山梨黑斑病的分子检测技术对该病害的早期及时防治及口岸检验检疫具有重要的意义.本研究通过分析比较砀山梨黑斑病菌与其他19种不同真菌的ITS序列,设计了1对特异性引物,能够从该病菌中特异性地扩增出1条398 bp的条带,而其他测试真菌中不能扩增出该条带.在灵敏度检测中,该菌的基因组DNA检出限为10 pg/μL,同时在发病的砀山梨果实组织样品和接种梨黑斑病菌的梨果实组织样品中检测出梨黑斑病菌.因此,可以将该对引物用于梨黑斑病早期诊断及病菌的PCR检测,为指导果农及时进行药剂防治提供有效的技术支撑.

为准确检测水稻白叶枯病菌、细菌性条斑病菌及这两种病菌的复合发生,利用软件DNAStar分析比较这两种菌的部分核酸序列,设计了检测这两种病菌的特异性引物.引物XooF-XooR能特异性扩增出水稻白叶枯病菌中一条大小162 bp的条带;引物XoocF1-XoocR1和XoocF2-XoocR2能够分别特异性扩增出水稻细菌性条斑病菌中690bp和945bp的条带.通过优化PCR反应条件,成功建立了多重PCR技术,可以对不同国家的水稻白叶枯病菌和细菌性条斑病菌进行准确检测,对由这两种病菌引起的复合侵染实现了准确诊断.

本文在综述澳大利亚鲜食葡萄产业现状的基础上，分析了其特点及面临的突出问题，提出中国出口农业提升内生动力和科技自主创新能力；加强政府宏观调控，发挥市场和行业协会的作用；强化质量意识，注重可持续发展的发展战略。

克隆并测定烟草炭疽菌rDNA全序列,序列全长2870 bp.序列比对发现,烟草炭疽菌与胶孢炭疽菌的rDNA序列相似性最高,达96.0％ ～ 96.2％;从构建的系统关系树也可以看出,烟草炭疽菌与胶孢炭疽菌聚成一个单独的分支,说明烟草炭疽菌与胶孢炭疽菌的亲缘关系最近.因此,结合烟草炭疽菌的形态学特征,可以初步将从贵州烟草分离的烟草炭疽菌鉴定为胶孢炭疽菌(Colletotrichum gloeosporioides).根据所测烟草炭疽菌的rDNA序列设计特异性引物,不仅可以从8种不同的烟草病原真菌中鉴定出烟草炭疽菌,而且可以从7种炭疽菌中鉴定出烟草炭疽菌.用烟草炭疽菌接种普通烟,以接种发病的病组织总DNA为模板,利用该特异性引物进行PCR扩增,同样可以扩增出特异性条带,表明该方法可用于烟草炭疽菌的鉴定和快速检测.

Aphelechoides besseyi is one of the most important nematodes on rice. In order to study the activity of methylene bisthiocyabate against rice nematodiasis, the field trails were conducted to test the efficacy of methylene bisthiocyabate in controlling this disease. The result demonstrated that,the control efficiency of 10%methylene bisthiocyabate EC and 95% cartap WP on stem tip nematode disease of rice were both above 84%, and that of 10% methylene bisthiocyabate EC was better. Morever, there was significant difference in rice yield treated by 10% methylene bisthiocyabate EC and 95% cartap WP, the rice yield treated with these two pesticides was significantly higher than that of CK.

Molecularly imprinted polymers(MIPs) with high selectivity to triazoles residues were prepared using triadimefon as template, trifluoromethyl acrylic acid (TFMAA) as functional monomer and ethylene glycol dimethacrylate (EGDMA) as cross. linker. The MIPs were applied as solid phase extraction materials. The optimum loading, washing and eluting conditions for molecularly imprinted solid phase extraction (MISPE) were established to obtain high selectivity and sensitivity. Coupled with LC-MS/MS, a high throughput and selective method was developed for simultaneous determination of 20 triazole pesticides in vegetables and fruits. The spiked recovery was investigated and the matrix effects were compared between MISPE and ENVI-Carb/PSA SPE columns. The results showed that the pesticide recoveries were in the range of 81.0% - 109.7% with RSD lower than 13% at 1.0, 2.0 and 10.0 mu g/kg spiked levels, and the matrix effect was in the range of 91.1% - 121.8%. Compared with ENVI-Carb/PSA SPE columns, MISPE exhibited selective binding properties for triazoles and the matrix effect was significantly decreased.

Fluxapyroxad,3-(difluoromethyl)-1-methyl-N-(3’,4’,5’-trifluorobiphenyl-2-yl) pyrazole-4-carboxamide (C18H12F5N3O, Fig. 1), is a SDHI fungicide, which is a new active ingredient that interferes with succinate ubiquinone reductase in the electron transport chain of fungi. Between 2008 and 2010, a total of 128 isolates of Rhizoctonia solani from Anhui Province of China were characterized for the baseline sensitivity to fluxapyroxad. The isolates collected between 2008 and 2010 all showed similar sensitivity to fluxapyroxad. Baseline sensitivity was distributed as unimodal curves with an average EC50 value of 0.054 ± 0.014 μg ml−1. However, EC50 values of boscalid for inhibition of mycelial growth of R. solani isolates ranged from 1.89 to 2.68 μg/ml and the average (±SE) EC50 value was 2.212 ± 0.228 μg/ml, indicating that the R. solani isolates were less sensitive to boscalid than that of fluxapyroxad. Fluxapyroxad exhibited excellent protective and curative activity against rice sheath blight and provided 82.6–94.2 % protective or curative control efficacy. In field trials, control efficacy of fluxapyroxad at 100 g a.i/ha 15 days and 30 day after second application was 83.4–88.0 %, suggesting excellent activity against sheath blight. Control efficacy of boscalid at a dosage of 600 g a.i/ha 15 days and 30 day after second application was about 51.7–57.0 %. There was a significant difference in the efficacy between fluxapyroxad and boscalid or jinggangmycin. These results suggested that fluxapyroxad is a good alternative fungicide to jinggangmycin for the control of rice sheath blight.