烟粉虱模式识别受体βGRPs的先天免疫应答

为探索烟粉虱的β-1,3-葡聚糖识别蛋白(BtβGRP)在烟粉虱先天免疫过程中的重要作用,对烟粉虱中鉴定的3个BtβGRPs基因编码的蛋白质进行结构分析,并与其它物种的23个同源蛋白进行系统进化分析,同时通过实时荧光定量PCR(quantitative real-time PCR,q RT-PCR)检测分析球孢白僵菌侵染烟粉虱不同虫态不同时间点后BtβGRPs的时间表达模式。结果显示:经鉴定3个BtβGRPs蛋白序列均含有βGRP家族特有的保守性结构域,即糖苷水解酶活性结构域;且3个BtβGRPs独自聚在进化树的一支上,可能参与烟粉虱特有的级联路径;3个BtβGRPs基因在不同虫态和不同诱导时间点与对照相比,除BtβGRP3在卵期未表达外,均有上调表达趋势,但诱导程度不同;成虫诱导24~36 h后达到峰值,比卵和若虫稍晚,可能是由于烟粉虱成虫体表有白色蜡粉等物质,延迟了球孢白僵菌对虫体的侵染。研究表明3个BtβGRPs蛋白可能参与了烟粉虱对真菌侵染的免疫反应,有可能成为烟粉虱生防的新靶标。     

Detection of airborne inoculum of Leptosphaeria maculans and Pyrenopeziza brassicae in oilseed rape crops by polymerase chain reaction (PCR) assays

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.     

应用聚合酶链式反应鉴定新疆棉花落叶型黄萎病菌

用一对棉花落叶型黄萎病菌的特异性引物D1和D2进行PCR扩增,对于落叶型黄萎病菌,该对引物可特异性地扩增产生一段550bp的产物,而非落叶型黄萎病菌则不能被扩增.供试的35个新疆黄萎菌系中,有3个菌系扩增出550bp大小的落叶型黄萎病菌特异性片段,表明目前新疆已存在落叶型黄萎病菌,用此技术可快速、准确地检疫和鉴定落叶型黄萎病菌.     

烟草线条病毒RT-PCR检测方法

根据已报道的烟草线条病毒外壳蛋白基因序列，设计出一对特异性引物，提取大豆病叶的总RNA，反转录成cDNA，PCR扩增出约400bp大小的产物，产物克隆到pGEM-T Easy载体上，转化大肠杆菌DH 5α，抽提的质粒用Not Ⅰ酶切，出现430bp大小的目标插入片段。经序列测定确认了插入片段的大小为404bp。     

枯草芽胞杆菌HMB19198在番茄叶片上定殖能力的分子检测

枯草芽胞杆菌HMB19198能有效防治番茄灰霉病,为快速、准确检测HMB19198在叶面的定殖能力,本研究通过对HMB19198全基因组序列比对分析,获得该菌株102 bp功能未知的独有基因序列,设计出针对HMB19198的特异性引物和探针。荧光定量PCR结果表明,该引物和探针对HMB19198具有较高的特异性,在有番茄叶片DNA干扰下,体系检测阈值为10²拷贝/μL。利用荧光定量PCR技术和菌落计数法检测了HMB19198在叶片上定殖动态。叶片喷施1×10⁸ cfu/mL的菌体悬浮液,0 d后菌体数量分别为1.7×10⁸拷贝/g叶片和8.9×10⁷ cfu/g叶片,2、4、6和8 d后菌株HMB19198在叶面的定殖数量逐渐降低,8 d后定殖数量分别为1.0×10⁷拷贝/g叶片和1.2×10⁷cfu/g叶片。防效试验结果表明,喷施菌株HMB19198悬浮液2 d后防效在80%以上,8 d后防效降为37.9%。     

Molecular detection of Fusarium solani f. sp. glycines in soybean roots and soil

A polymerase chain reaction (PCR)-based method was developed to detect DNA of Fusarium solani f. sp. glycines , the cause of soybean sudden death syndrome. Two pairs of primers, Fsg1/Fsg2 designed from the mitochondrial small subunit ribosomal RNA gene, and FsgEF1/FsgEF2 designed from the translation elongation factor 1-α gene, produced PCR products of 438 and 237 bp, respectively. Primer specificity was tested with DNA from 82 F. solani f. sp. glycines , 55 F. solani non-SDS isolates, 43 isolates of 17 soybean fungal pathogens and the oomycete Phytophthora sojae , and soybean. The sensitivity of primer Fsg1/Fsg2 was 10 pg while that of FsgEF1/FsgEF2 was 1 ng when using F. solani f. sp. glycines total genomic DNA or down to 10³ macroconidia g⁻¹ soil. Nested PCR increased the sensitivity of the PCR assay 1000-fold to 10 fg using primers Fsg1/Fsg2, and 1 pg using primers FsgEF1/FsgEF2. F. solani f. sp. glycines DNA was detected in field-grown soybean roots and soil by PCR using either single pairs of primers or the combination of two pairs of primers. The occurrence of F. solani f. sp. glycines was determined using nested PCR for 47 soil samples collected from soybean fields in 20 counties of Illinois in 1999. F. solani f. sp. glycines was detected in soil samples from all five Illinois Agricultural Statistic Districts including 100, 89, 50, 92 and 50% of the samples from East, Central, North-east and West Districts, respectively.     

Pathogenesis-related gene expression in rice is correlated with developmentally controlled Xa21-mediated resistance against Xanthomonas oryzae pv. oryzae

Disease resistance mediated by the resistance gene Xa21 is developmentally controlled in rice. We examined the relationship between Pathogenesis Related (PR) defense gene expression and Xa21-mediated developmental disease resistance induced by Xanthomonas oryzae pv. oryzae (Xoo). OsPR1a, OsPR1b, and OsPR1c genes were cloned and their induction was analyzed, in addition to the OsPR10a gene, at the juvenile and adult stages in response to a wildtype Xoo strain that induces a resistance response (incompatible interaction) and an isogenic mutant Xoo strain that does not (compatible interaction). We found that the adult stage leaves are more competent to express these OsPR1 genes and that the Xa21 locus is required for the highest levels of induction.     

‘砀山酥梨’褐皮芽变木质素含量及相关酶活性与CCoAOMT 表达量分析

 为了探讨‘砀山酥梨’芽变品系‘锈酥’果皮褐色形成机理,采用分光光度法测定盛花后25、50、75、100、125、150和175 d果皮中木质素含量和相关酶活性变化;从构建的‘锈酥’正向SSH-cDNA文库中筛选出与木质素生物合成密切相关的CCoAOMT-EST,通过实时荧光定量PCR测定了‘砀山酥梨’和‘锈酥’果皮中CCoAOMT的相对表达量。结果表明：‘锈酥’果皮发育前期木质素增量较大,且木质素增量累计比‘砀山酥梨’高12.2%;‘砀山酥梨’和‘锈酥’果皮中PAL、4CL、CAD酶活性均在花后75 d达到最大值,而POD酶活性则在花后125 d出现高峰;二者果皮中4种酶活性变化趋势基本一致,但‘锈酥’果皮中均相对较高;‘锈酥’果皮中的PAL和4CL酶活性与木质素增量变化均呈显著正相关,而‘砀山酥梨’则未呈现出此规律;在果实生长发育各个时期,‘锈酥’果皮中CCoAOMT相对表达量均高于‘砀山酥梨’。因此推测,‘锈酥’果皮褐色形成与果皮中木质素积累及相关酶活性提高有关,果皮中CCoAOMT的增量表达是‘锈酥’果实褐皮形成的重要原因之一。     

湖南柑橘衰退病调查分析及弱毒系筛选

 The investigation showed that stem-pitting Citrus tristeza virus (CTV)occurred commonly in citrus production areas in several varieties of Hunan Province. Accurate detection of CTV strains was performed by p23/PCR method, PCR and the results indicated that the most samples were infected with several CTV isolates. Three mild strains were isolated and their pathogenicity was identified by biological identification, it indicated that p23/PCR groups had uniformity with the pathogenicity of CTV isolates. Furthermore, three mild isolates were tested in the cross protection by analysis of biological symptoms and composition of p23 gene. Different protecting effects were observed among these strains and W17 mild isolate was effective.     

蔬菜保护地木霉菌rDNA-ITS序列和UP-PCR遗传多样性分析

采用传统形态学分类和ITS序列比对的方法,研究蔬菜保护地土壤中木霉菌种群分布和遗传多样性。木霉菌分离培养结果显示,共获得397株木霉菌,鉴定出11个种,分别为:长枝木霉Trichoderma longibrachiatum、深绿木霉T.atroviride、哈茨木霉T.harzianum、粘绿木霉T.viren、微孢木霉T.minutisporum、拟康木霉T.pseudokoningii、黄绿木霉T.aureoviride、非钩木霉T.inhamatum、棘孢木霉T.asperellum、长孢木霉T.longipile和螺旋木霉T.helicum。经ITS序列建立系统发育树后,将木霉菌分为5个组。用5条通用引物经UP-PCR扩增后,扩增出46条谱带,其中多态性条带43条,占总条带数的93.5%。遗传多样性分析表明,当相似系数为0.80时,可将24个菌株划分为9个组。UP-PCR与ITS序列相比,更能体现木霉菌种间和种内的亲缘关系及遗传差异性,可以作为木霉菌分类的辅助方法。