Epidemiology of dark leaf spot caused by Alternaria brassicicola and A. brassicae in organic seed production of cauliflower

In organic seed production of Brassica vegetables, infections by Alternaria brassicicola and A. brassicae can cause severe losses of yield and seed quality. Four field experiments with or without artificial inoculation with A. brassicicola were conducted in organically managed seed‐production crops of cauliflower cv. Opaal RZ in 2005 and 2006 in the Netherlands. The development of A. brassicicola and A. brassicae on pod tissues and developing seeds was followed and seed quality was assessed. Alternaria brassicicola was externally present on 1·2% of the seeds 14 days after flowering and observed internally within 4 weeks after flowering. In both seasons, seed colonization by the pathogen increased slowly until maturation but sharply increased during maturation. A similar pattern was found for the colonization of pod tissues by A. brassicicola as quantified by TaqMan‐PCR. The incidence of A. brassicicola on mature seeds reached 70–90%. Internal colonization was found for 62–80% of the seeds. External and internal seed colonization by A. brassicae was much lower, with incidences below 3%. The quality of harvested seeds was generally low, with less than 80% of seeds able to germinate. Seed quality was not affected by warm water treatments. It was concluded that A. brassicicola and A. brassicae have the potential to infect pods and seeds soon after flowering. For the production of high quality seeds, producers must prevent such early infections. Therefore, new control measures are needed for use in organic cropping systems.     

喀斯特炭疽菌TaqMan探针实时荧光PCR检测方法的建立

为寻求喀斯特炭疽菌的快速检测方法,利用TaqMan探针实时荧光定量PCR技术,以喀斯特炭疽菌基因组中的ACT基因为靶序列设计并筛选特异性引物探针,通过特异性、灵敏度、重复性试验建立喀斯特炭疽菌TaqMan探针实时荧光PCR检测方法。结果表明:建立方法的特异性较强、灵敏度较高、重复性稳定性较好,最低检测限为0.2pg DNA/反应,标准曲线的线性关系良好,相关系数为0.998。该方法操作简便,可用于进出境口岸喀斯特炭疽菌的检疫。     

A DNA Probe for Rapid Screening of Sugar Beet (Beta vulgaris L.) Carrying Extra Chromosomes from Wild Beets of the Procumbentes Section

Genes from wild species of the Procumbentes section can be transferred to sugar beet chromosomes via translocations. Since large translocations, including for example a gene for nematode resistance, generally result in meiotic disturbances, there is a need to select new diploid resistant beets from progenies of monosomic wild beet addition lines. A dispersed repetitive DNA probe, which is closely correlated with the resistance gene and hybridizes exclusively with wild beet DNA, appears to be highly superior to selection based on isozyme markers. Characteristic ‘fingerprints’ on the available monosomic addition lines reveal the existence of at last 5 different chromosomes in the wild species each housing a gene for nematode resistance. This probe can be used advantageously to identify individuals carrying the intact or fragmented wild beet chromosomes, or even various amounts of translocated chromatin. Strategies are discussed for the identification of new translocation types using straightforward squash dot or Southern hybridization techniques in combination with the wild beet DNA probe.     

实时荧光定量PCR法检测对虾皮下和造血器官坏死病毒

应用实时荧光定量PCR最常用的TaqMan探针技术设计了探针和引物,并且用质粒技术构建了含有传染性皮下及造血器官坏死病毒(IHHNV)基因片段的阳性质控品,建立了检测IHHNVV的实时荧光定量PCR方法。对影响PCR的主要因素进行了优化,Mg2+浓度、引物和探针浓度、退火温度等均对扩增效率有明显影响。当Mg2+浓度为3.0-4.5mmol,退火温度为59～60℃时可获得最佳扩增效果。灵敏性试验表明该反应可检测体系中10拷贝的病毒核酸;该体系检测IHHNV具有很高特异性;对临床样品检测结果表明,该方法能快速、准确地检测样品中的IHHNV。     

H3亚型猪流感病毒荧光定量PCR检测方法的建立

通过RT-PCR方法克隆了H3亚型猪流感病毒HA基因一段靶序列,构建重组质粒作为标准阳性模板.根据GenBank中的H3亚型猪流感病毒HA基因保守序列设计了用于FQ-PCR的1对引物和1条TaqMan探针.通过条件优化,以10倍系列稀释的质粒为标准品进行荧光定量PCR扩增,并制作标准曲线,建立了检测H3亚型猪流感的荧光定量PCR方法.结果表明,该方法检测灵敏度可达1.0×100拷贝/μL,线性范围为109～100,达10个数量级;对起始浓度为1.0×109、1.0×108、1.0×107拷贝/μL的标准品的最终实际测得值(Ct)分别为13.68,18.21和20.57;变异系数分别为0.31%、0.17%和0.12%,均小于5%,说明此方法具有良好的准确性和重现性.对阳性组织病料的检测表明,该方法的检测灵敏度高出常规PCR,与套式PCR具有相近的灵敏度.     

荧光定量PCR技术及其应用研究进展

荧光定量PCR是近年发展起来的一种新的实时定量检测特定核酸技术,它是核酸探针技术、荧光共振能量传递技术和PCR技术的有机结合,而荧光探针是荧光定量PCR的核心.荧光定量PCR具有特异性强、灵敏度高、重复性好、定量准确、速度快、全封闭反应争特点,是对原有PCR技术的革新,扩大了PCR的应用范围.论文综述了FQ-PCR技术的原理、FQ-PCR实时定量检测系统及其应用.     

基于TaqMan MGB探针的可可花瘿病菌快速检测方法

可可花瘿病菌是一种我国进境植物检疫性真菌?本文根据可可花瘿病菌EF1α基因的保守序列, 设计并合成1对特异性的实时荧光PCR引物和1条TaqMan MGB探针, 建立了可可花瘿病菌的实时荧光PCR检测方法?特异性试验结果表明, 该检测方法能够特异性检出可可花瘿病菌; 实时荧光PCR优化反应条件为引物终浓度0.2 μmol/L, 探针终浓度0.6 μmol/L; 灵敏度试验结果表明, 20 μL反应体系中可可花瘿病菌DNA含量最低检测限为10 pg; 重复性试验结果表明, 该检测方法的重复性和稳定性良好; 接种试验样品检测结果表明, 该方法可用于疑似携带可可花瘿病菌样品的检测与初筛?本文建立的方法具有良好的灵敏性?特异性和应用性, 为可可花瘿病菌早期快速检测提供了一种有效手段?     

Errors in repeated measurements of soil water content in pots using a ThetaProbe

Abstract. The accuracy of a ThetaProbe (Delta-T Devices Ltd, UK) to obtain repeated measures of soil water content in pot plants was tested. This alternative to balance determinations led to a large underestimation of water content, varying from 12.2 to 21.8% of the total water content, depending on soil type.     

Characterization of the goldfish fecal microflora by the fluorescent in situ hybridization method

ABSTRACT:   Bacterial populations in goldfish feces were characterized by the fluorescent in situ hybridization (FISH) method. A total of nine different group-specific rRNA-targeted oligonucleotide probes were used. Approximately half of the microbial cells (57.8 ± 16.7%) were detected with a probe EUB338 and found to be bacteria. The microbial cells in 33–35 of the 35 samples from five specimens strongly hybridized with probes ALF1b, BET42a and GAM42a, suggesting that goldfish intestinal bacteria are mainly composed of α, β and γ-subclasses of Proteobacteria. The fact that a probe AER66 reacted with 25.6 ± 14.2% of the total microbial cells in all 35 samples, demonstrated that genus Aeromonas was the dominant species in the goldfish intestines. Genus Bacteroides including Bacteroides type A detected with a probe BAC303 was observed in 15 of 35 samples while other taxonomic groups determined with HGC69a, CF39a and P72 were detected in 6–11 of 35 samples. These results strongly suggest that Bacteroides shows the greatest daily fluctuation and interindividual variation in the intestines of goldfish. Moreover, the FISH method was proven to be useful for rapid enumeration of taxonomic groups of fish intestinal bacteria.