西瓜细菌性果斑病菌快速免疫PCR检测

对种子携带的西瓜细菌性果斑病菌(Acidovorax avenae subsp. citrulli)进行简单抽提和纯化,不经过DNA提取而以病原菌为模板进行PCR检测.结果显示,对带菌种子提取液采用直接PCR法最低检出限为3600个细菌/mL,而免疫PCR最低检出限可达到600个细菌/mL.免疫PCR法可以有效富集病原后再扩增,方便快速,成本低,灵敏度高,适用于种子携带微量的西瓜细菌性果斑病菌的快速鉴定.     

甘肃省西瓜细菌性果斑病的诊断

对从甘肃采集到的疑似西瓜果斑病的病瓜及种子中分离获得的菌株,通过形态学特征、革兰氏染色、致病性测定以及利用美国Agdia公司的专化型免疫凝聚试剂条测定和16S rDNA分子生物学方法进行了诊断鉴定,结果表明,该菌株符合燕麦噬酸菌西瓜亚种(Acidovorax avenae subsp. citrulli)的特性,因此,确定甘肃省已发生西瓜果斑病,结果为甘肃省有效控制此病提供了依据。     

瓜类细菌性果斑病菌对硫酸铜的敏感性检测与分析

果斑病菌两个亚群的143个菌株继代培养10代后,通过平板画线法对各菌株的硫酸铜敏感性检测结果表明：在硫酸铜浓度为300 μg/mL(1.88 mmol/L)时只有一株菌株表现敏感,在浓度达到750 μg/mL(4.69 mmol/L)时所有菌株均表现敏感;浓度为450~650 μg/mL (2.81~4.06 mmol/L)时分离自甜瓜的敏感菌株所占比率显著低于西瓜菌株;继代培养后所有菌株的硫酸铜敏感性没有改变,证实果斑病菌具有稳定的抗铜性。     

免疫凝聚试纸条和TaqMan探针实时荧光PCR检测西瓜细菌性果斑病菌比较研究

 以西瓜细菌性果斑病菌(Acidovorax avenae subsp.citrulli)菌悬液和田间采集的病组织为试材,研究了免疫凝聚试纸条和实时荧光PCR技术检测的灵敏度和适应性。结果表明,免疫凝聚试纸条检测灵敏度为10⁶ cfu/mL,具有简便、快速、易操作特点,适用于田间快速检测和病害诊断;TaqMan探针实时荧光PCR检测灵敏度达10^3~4 cfu/mL,比传统PCR检测灵敏度(10⁵ cfu/mL)提高了10~100倍,且不需要琼脂糖凝胶电泳、溴化乙锭染色和Southern杂交。但需要昂贵的仪器和试剂,适用于室内检测及相关研究。     

应用蛋白宏阵列快速检测西瓜细菌性果斑病菌

蛋白阵列(Protein array)也叫蛋白芯片,是生物芯片(Biochip)的一种,主要利用抗原/抗体可特异性结合原理制备而成。按阵列样点大小,蛋白阵列又可分为微阵列(Microarray)和宏阵列(Mac-roarray)两类。微阵列集成度极高,可进行高通量     

天津滨海新区泡桐丛枝病植原体16S rDNA基因序列分析

利用分子生物学技术对天津滨海新区泡桐丛枝病病原进行分类鉴定。采用植原体16S rDNA通用引物R16mF2/R16mR1对患病植株总DNA进行PCR扩增,得到约1.4 kb特异性片段。克隆测序、Blast比对和iPhyClassifier分析结果表明,天津滨海新区泡桐丛枝植原体16S rDNA基因片段长1 432 bp,与国内泡桐丛枝植原体PY株系相似性最高,达99.86%,归属于16SrI组(aster yellows group,翠菊黄化组)D亚组。系统树构建与分析显示,泡桐丛枝病天津滨海株PaWB-TJBH与16SrI其他亚组亲缘关系较近,同在16SrI组进化枝上,与16Sr I-D组亲缘关系最近;16S rDNA序列RFLP电子酶切图谱表明,PaWB-TJBH属于16SrI-D组一个成员,与同源性比较和系统进化分析结果一致。     

种子引发处理对无籽西瓜幼苗生长的影响和对细菌性果斑病菌消毒的效果

 种子出苗率低和传带细菌性果斑病菌是制约三倍体无籽西瓜生产的主要原因。本试验利用KMnO₄、CuSO₄和ZnSO₄的不同浓度溶液对无籽西瓜种子进行不同时间引发处理, 通过滤纸发芽试验, 观测其对种子发芽和幼苗生长的促进作用;借助平板法测定引发溶液对细菌性果斑病菌FC247的抑制作用, 免疫凝聚试纸条和传统PCR检测引发处理对种子人工接菌FC247的消毒效果。结果表明:0.1% CuSO₄溶液引发处理4 h和0.2% ZnSO₄溶液引发处理24 h, 分别比未引发处理的无籽西瓜种子发芽率提高71.1%和73.3%, 并能显著提高发芽整齐度和幼苗素质;同时, 引发溶液对细菌性果斑病菌有显著抑制作用, 并对人工接菌种子表现出-定程度的消毒效果。     

海南长春花黄化病植原体的16S rDNA序列分析研究

 Periwinkle(Catharanthus roseus) yellows is a common disease in Hainan. Periwinkle's leaf tissue with symptoms was assayed for phytoplasma infection by using PCR assay employing phytoplasma universal 16S rRNA gene primers (Rl6mF2/Rl6mR1). A PCR product (about 1.4 kb) was amplified from periwinkle showed yellows. Nucleotide sequencing and phylogenetic tree analysis showed that the amplified 16S rDNA contained 1 432 nucleotides, the most homology was 98.1% with the members of elm yellows group (16S r Ⅴ) and clustered in the same clade, while it was under 96.1% with other phytoplasma groups. Our results suggested that the phytoplasma sample belonged to 16S rⅤgroup and was tentatively named as Hainan periwinkle yellows phytoplasma (PY-Hn). This is the first report of existence of 16S r Ⅴ group phytoplasma in naturally infected periwinkle.     

仙人掌丛枝病植原体检测和16S rDNA片段序列分析

 对仙人掌丛生幼嫩组织进行超薄切片电镜观察,在韧皮部筛管中存在大量植原体;根据植原体16S rRNA基因保守序列设计的通用引物对R16 F₂/R₂,应用PCR技术对仙人掌丛枝病进行分子检测,结果扩增到约1.2 kb的特异性片段,而在健康组织中却没有此特异片段;通过16S rDNA片段核酸序列同源性比较,结果表明仙人掌丛枝病植原体与花生丛枝病植原体亲缘关系最近,据此可初步判断仙人掌丛枝病植原体是一种属于16Sr Ⅱ组的植原体,基本确定了其分类地位。     

来源于湖北省枣疯病植原体分离物16S rDNA和rp基因的序列分析

以田间采集的来源于我国湖北省枣树产业主产区随州市随县种植的表现为"枣疯病"症状的枣树分离株为试材,对其16S rDNA和核糖体蛋白(ribosomal protein,rp)基因采用Nested-PCR进行扩增以及序列分析。结果表明,湖北JWB-Hubei植原体分离物16S rDNA基因的核苷酸序列与我国山东、河南等地的分离株一致率均为99%以上,在进化树中位于同一亚组的不同进化分支;虚拟RFLP图谱分析表明,JWB-Hubei属于16SrV-B亚组一个成员,与其进化树分组结果一致。JWBHubei分离株rp基因的核苷酸序列也与我国山东、陕西等地区的分离株一致率均为99%以上,在进化树中聚为同一亚组,与报道的基于RFLP分类属于rpV-C亚组的中国枣疯病分离物(JWB)聚集于同一亚组不同分支。该研究结果明确了湖北省枣疯病植原体的分类地位以及与来源于我国不同地区枣疯病分离株之间的遗传进化关系,为进一步研究植原体的株系划分、基因遗传变异研究提供了理论基础。     

Phylogenetic analysis of the citrus Huanglongbing (HLB) bacterium based on the sequences of 16S rDNA and 16S/23S rDNA intergenic regions among isolates in China

Phylogenetic analysis of Chinese isolates of the citrus Huanglongbing (HLB) bacterium based on the 16S rDNA and 16S/23S rDNA intergenic regions sequences was carried out. Nine HLB samples collected from different hosts with different symptoms in seven Chinese provinces, were subjected to PCR for amplifying and sequencing the 16S rDNA. The identity level among Chinese isolates was 98.5% to 100% and was the same with the Indian HLB isolate ‘Poona’ (GenBank accession number: L22532). By contrast, identity values were 97.5% to 97.8% with Candidatus Liberibacter africanus strain ‘Nelspruit’ (L22533), 96.3% to 97.3% with Ca. L. africanus subsp. ‘Capensis’ (AF137368), 95.3% to 96.5% with the Ca. Liberibacter sp. ‘LSg2’ (AY919312), and 94.9% to 96.0% with a strain of Ca. L. americanus from Brazil (São Paulo State; AY742824). A phylogenetic tree constructed with 16S rDNA sequences showed that all Chinese isolates belong to Ca. L. asiaticum. Analysis of the 16S/23S rDNA intergenic region was conducted on 18 HLB-diseased citrus samples with different symptoms, collected in seven provinces. These isolates showed no obvious variation and had an identity level >99.0% with one another. Sequence analysis of 16S/23S rDNA intergenic region and the relative phylogenetic tree showed that the Chinese isolates are very close to Ca. L. asiaticus, and distinct from Ca. L. africanus and Ca. L. americanus. These results suggest that the Chinese HLB isolates belong to the species Candidatus Liberibacter asiaticus. This is the first report on the classification of HLB isolates from China based on molecular investigations.     

哈密瓜细菌性果斑病菌群体感应系统的检测

本文利用群体感应信号报告菌Agrobacterium tumefaciens NTL4(pZLR4),在LB报告平板上对燕麦食酸菌西瓜亚种的19个菌株进行了初步检测,发现18个菌株有群体感应信号产生.用琼脂条法对菌株Pslb-94进一步检测,证实菌株Pslb-94存在群体感应系统.提取了该菌株信号物质,反相薄层层析(TLC)检测证明该菌株能产生群体感应信号分子.     

哈蜜瓜种子带细菌性果斑病菌检测技术的研究

哈蜜瓜细菌性果斑病是世界性的检疫对象之一,主要以种子带菌进行远距离传播,本文通过温室育苗、室内分离和PCR(聚合酶链式反应)技术对哈蜜瓜种子带菌情况和主要带菌部位进行了检验,结果表明:3种方法都可以对种子带菌情况进行检测,分离检测和PCR检测还可以确定种子的带菌部位,PCR技术具有特异性强、灵敏度高、检测时间短等特点.另外检测结果也表明种子带菌以种皮为主,种仁的带菌量较少.     

利用GICA-PCR快速检测瓜类果斑病菌

瓜类果斑病菌(Acidovorax avenae subsp.citrulli,Aac)是瓜类作物上重要的病原细菌,为我国进境植物检疫性有害生物。胶体金免疫层析试纸条方便快捷,应用广泛。该方法使用不当会出现假阳性问题,仅适用于病原菌的初筛检测。本研究将Aac胶体金免疫层析方法(GICA)与PCR技术相结合,建立了GICA-PCR检测方法。检测结果表明,该方法在蛋白与核酸2个层面上从发病西瓜叶片上检测到瓜类果斑病菌,有效解决了试纸条检测的假阳性问题,提高了瓜类果斑病菌检测的准确性,值得推广应用。     

西瓜细菌性果斑病研究进展

西瓜细菌性果斑病是由类产碱假单胞菌西瓜亚种〔Pseudomonas pseudoalcaligenes subsp. citrulli (Schaad et al. 1978)〕所引起，1992年该病菌改名为燕麦食酸菌西瓜亚种〔Acidovorax avenae subsp. citrulli (Willems et al. 1992) 〕。革兰氏阴性菌，属rRNA组I。不产生荧光和其他色素，单根极生鞭毛，严格好氧。不产生精氨酸水解酶，能在41℃下生长，但不能在4℃生长，明胶液化力弱，氧化酶和2－酮葡糖酸试验阳性。利用葡萄糖和蔗糖作碳源结果不一致；但利用β－丙氨酸、柠檬酸盐、乙醇、乙醇胺、果糖、L－亮氨酸和D－丝氨酸结果一致…     

Molecular identification and prevalence of Acidovorax avenae subsp. avenae causing red stripe of sugarcane in China

Red stripe caused by the bacterium Acidovorax avenae subsp. avenae (Aaa) is a disease of sugarcane that is distributed worldwide. In this study, 108 sugarcane leaf samples were collected in 2013–2016 from nine sugarcane‐growing regions in China. Aaa was detected by PCR with specific and novel primers from the 16S–23S rDNA internal transcribed spacer region in 81 of 84 (96%) leaves with red stripe symptoms and in 20 of 24 (83%) leaves without symptoms. Furthermore, Aaa was detected in all nine sampling locations representing six sugarcane‐producing provinces in China. The 101 amplified fragments were cloned and sequenced. The size of the nucleotide sequences varied from 436 to 454 bp and the sequence identity ranged from 89.2% to 100%, suggesting a significant genetic variation among Aaa strains from China. Five major restriction fragment length polymorphism (RFLP) profiles were obtained by in silico and polyacrylamide gel electrophoresis analyses of the PCR products digested with HindIII and EcoRI. The causal agent of sugarcane red stripe was also successfully isolated from a diseased plant and its pathogenicity confirmed by inoculation of healthy sugarcane plantlets and reproduction of disease symptoms. The data showed that Aaa is currently widespread in China, suggesting that control methods should be implemented to limit the impact of red stripe on sugarcane production.     

12种寄主来源的茄科雷尔氏菌16S-23SrDNA间隔区序列比较

应用PCR方法,获得了分离自广东番茄、茄子、辣椒、烟草、空心菜、沙姜、姜、马铃薯、花生、菊花、桑树和藿香等12种作物21个茄科雷尔氏菌菌株的16S 23S rDNA 间隔区序列（ITS）。序列分析结果表明,除HZ 1菌株外,其余20个茄科雷尔氏菌菌株ITS序列长均为503 bp,序列间相似性99.2%~100%,序列间差异仅1~4 bp;而HZ 1菌株的ITS序列长为498 bp,与其他菌株的ITS序列相似性为95.4%~95.6%。这些结果说明,这21株来源于12种不同寄主的茄科雷尔氏菌菌株的16S 23S rDNA ITS序列比较保守。系统进化分析显示,仅菌株HZ 1聚类于茄科雷尔氏菌区组2中,其余20个菌株均聚类于茄科雷尔氏菌区组1中。