一株新的胡萝卜软腐欧文氏菌的分离和鉴定

从大白菜软腐组织中分离出一株软腐病细菌BC1,经过形态观察、生理生化特性分析、致病性检测和16S rDNA序列分析,该分离物被鉴定为胡萝卜软腐欧文氏菌胡萝卜亚种（Erwinia carotovora subsp. carotovora, Ecc）的一个新菌株,编号为BC1。这是首次从16S rDNA序列水平上对在我国分布的软腐欧文氏菌进行鉴定。Ecc BC1的16S rDNA序列与其它软腐欧文氏菌株的16S rDNA序列之间同源性达987%～993%,而且在系统发育树中独立于Ecc其它菌株。序列分析结果表明,Ecc BC1具有至少2种不同的16S rDNA序列,它们都在第459位和473位（相对于大肠杆菌16S rDNA序列）发生碱基突变,同一基因中两个突变位点之间彼此互补,处于16S rRNA螺旋H17颈部,而且这两处碱基变异只存在于BC1菌株中。通过与其它软腐欧文氏菌亚种和菌株16S rDNA序列进行比对分析,还进一步鉴定出一些BC1菌株特异的16S rDNA碱基突变位点。本文报道的Ecc BC1两个16S rDNA序列在GenBank中的登录号分别为AY309068和AY309069。     

拟南芥对细菌性软腐病抗性变异分析

由胡萝卜软腐欧文氏菌胡萝卜软腐亚种 Erwinia carotovora subsp.carotovora （Ecc）引起的细菌性软腐病是世界性的重要流行病害,由于缺乏天然抗源,研究进展缓慢,拟南芥成为软腐病抗性研究的主要试材。本文选用 29 份拟南芥材料,制定了病情分级标准,以接种后 48 小时的病情指数作为软腐病抗性的鉴定指标,筛选出抗软腐病材料 CS906 和感病材料 CS20。通过分析抗、感材料接种 Ecc 后 AOS、ERF1-1、PR1、PDF1.2 和 PAL1 等 5 个基因的表达变化,发现 SA、ET 和 MJ 信号途径都参与了拟南芥对 Ecc 的防卫反应,且基因表达模式在抗、感材料中相似,差异体现在表达量上。本研究对深入探讨拟南芥软腐病抗病机制具有重要意义。     

魔芋软腐病菌分子鉴定与遗传多样性

通过对分离的魔芋软腐病菌株和其它参试菌株的致病性测定、选择性培养基培养性状观察和16S-23S rDNA转录间隔区PCR(ITS-PCR)分析,将测试的33株软腐病菌株主要分为3个组群。第1组群为胡萝卜软腐欧文氏杆菌胡萝卜软腐亚种(Erwinia carotovorasubsp.carotovora,E.c.c.);第2组群为菊欧文氏杆菌(Erwinia chrysanthemi,E.ch.);还有一组未能确定的菌株。利用细菌基因组重复序列通用引物BOX和J3进行Rep-PCR特异性扩增,引起软腐病的菌株E.c.c.和E.ch.(ITS-PCR鉴定)种内的Rep-PCR指纹存在明显的遗传分化,经聚类分析,在0.1水平上把E.c.c.13株区分为5个类群。     

几种野生植物提取物抑菌作用研究

本文以乙醇为溶剂,分别提取了瓦松、泽漆及一组中草药配方的有效成分;室内测定了三种提取液对5种植物病原菌(棉花枯萎病菌Fusarium oxysporum f.sp.vasinfectum;小麦赤霉病菌Fusarium graminearum Schw;西瓜枯萎病菌Fusarium oxysporum f.sp.niveum;仙人掌软腐病菌Erwinia chrysanthemi pv.chrysanthemi;魔芋软腐病菌Erwiniacarotovora pv.carotovora)的抑菌作用.结果表明:三种提取物对镰刀菌均有不同程度的抑制作用,且三种提取物的抑菌效果差异显著,其中瓦松的抑菌效果最强.当在50 mL培养基中加入5mL不同提取液时,瓦松对不同镰刀菌的相对抑制率为91.39%～100%;中草药配方提取液对不同镰刀菌的相对抑制率分别为41.11%～85.37%;而泽漆提取液对镰刀菌的相对抑制率仅为12.86%～21.23%;当在50 mL培养基中加入提取液体积降低为2 mL、1 mL、0.5mL时,三种提取液对三种镰刀菌的抑制效果骤然下降,但下降的梯度不一.对大白菜软腐病菌与魔芋软腐病菌而言也是瓦松的抑菌效果最强,中草药配方次之,泽漆的抑制效果最差.     

新疆石河子地区棉花黄萎病菌分离鉴定及其致病力分析

旨在探究新疆石河子地区棉花黄萎病菌遗传变异及致病力分化情况。通过对分离菌株显微观察、ITS序列克隆及分子进化树的构建,对来源不同的棉花黄萎病菌分离鉴定以及致病力进行分析研究。结果表明:分离得到的24株棉花黄萎菌均属大丽轮枝菌(Verticillium dahliae),其中11株菌株为落叶型棉花黄萎菌,其余为非落叶型棉花黄萎菌;分子进化树结果显示24株菌在系统进化方式上属于2个不同的分支;相同寄主的致病力结果显示24株菌的致病力分为强中弱3个类型,其中强致病力比例为75%,相比2010年的51.7%提高了23.3%,表明石河子地区棉花黄萎菌群体致病力类型发生较大变化,强致病力菌系的比例增加,这或许与石河子地区常年种植棉花有一定的关系;其中SHZ-9为致病力最强的菌系,而SHZ-4为致病力最弱菌系,表明石河子地区黄萎菌存在着明显的致病力分化现象;不同致病力菌株按照营养亲和性可分为3个营养亲和群(VCGs)。本研究结果表明新疆石河子地区棉花黄萎病的致病菌基本是大丽轮枝菌,强致病力菌系占的比例在增加,且菌株在遗传分化上存在明显差异。     

粤、闽、贵烟草青枯病菌分离株及其致病力

【目的】从粤、闽、贵3地烟草青枯病株中分离青枯病菌,测定其致病力并探讨判断致病力的方法。【方法】采用氯化三苯基四氮唑(简称TTC)培养基和蛋白质电泳相结合对青枯病菌致病力进行测定,并分析菌株生化型。【结果】已分离出烟草青枯病菌59株,依据致病力进行划分,无致病力菌株5株,有致病力菌株54株。不同致病力菌株蛋白电泳特定条带存在差异。粤、闽、贵3地烟草青枯病菌有致病力菌株均占主导地位,广东有致病力菌株比例略大,福建次之,贵州最小。按生化型划分,59株青枯病菌中1株属于生化型Ⅳ外,其他58株属于生化型Ⅲ或其亚型生化型Ⅲ-1。【结论】烟草青枯病菌致病力存在差异,SDS-PAGE蛋白指纹中的特定条带可作为判定青枯病菌有无致病力的依据。     

EcbCSL101菌株hrpN基因的克隆、表达及HarpinEcbCSL101蛋白的生物学活性

胡萝卜软腐欧文氏菌甜菜亚种(Erwinia carotovora subsp. betavasculorum) EcbCSL101菌株具有很强胞外酶分泌活性, 接种非寄主植物烟草引起过敏反应。Southern blotting结果表明EcbCSL101菌株中含有hrpN 基因。PCR扩增含EcbCSL101完整开放阅读框的DNA片段并克隆到表达载体pET28a(+)中。核苷酸序列分析表明, EcbCSL101菌株的hrpN 基因的ORF为1113 bp, 编码36.65 kD HarpinEcbCSL101蛋白(GenBank, DQ355519),与其它几种软腐欧文氏菌Harpin蛋白有较高的同源性。将含有hrpNEcbCSL101基因的重组质粒转化到大肠杆菌JM109(DE3)中进行表达,纯化后的HarpinEcbCSL101能诱导烟草发生过敏反应。     

江西稻瘟病菌稻巨座壳致病性分化年度动态变化

为明确江西水稻种植区稻巨座壳菌（稻瘟病病菌）的年际变化规律,本研究采用7个中国鉴别寄主与30个单基因鉴别品系两套鉴别寄主分别鉴定分析了2006-2018年间从江西37个水稻主要种植县市分离的1 161个稻瘟病单孢菌的生理小种、致病力、致病类型与无毒基因型等。研究结果表明,江西稻瘟病菌可以分成7群49个生理小种,其中ZA、ZB、ZC群为优势种群,ZB13为优势小种,出现频率为18.00%,以毒性较强的强致病力菌株为主;江西稻瘟病菌在生理小种构成、优势小种、致病力年际变化方面均具有3-5年的周期性;江西历年稻瘟病菌的致病类型较为丰富且存在年度差异,菌株致病类型占各年度总菌株数的82.79%-98.21%,优势致病型菌株占当年总菌株的3.57%-5.77%;历年稻瘟病菌的无毒基因个数为24-29个,其中Avr-Pizt、Avr-Piz5、Avr-Pik、Avr-Pik(C)在历年供试菌株中出现频率较高,说明与之相对应的抗瘟基因在江西抗病育种与抗性品种布局中具有较好的应用价值。     

广东省水稻纹枯病菌遗传多样性与致病力分化的研究

利用随机引物扩增多态性DNA（PAPD）分子标记技术分析了来自广东省7个县市48个水稻纹枯病菌菌株的遗传多样性,以筛选出的10个随机引物对菌株进行PAPD－PCR扩增,共产生了98个PAPD分子标记,其中89．9％的片段具有多态性。48菌株间的遗传相似性系数（以Nei基因一致度表示）在0．56－0．949之间,用UPGMA和类分析可将它们分为5个RAPD遗传聚类群（A、B、C、D、E）,相同地区来源的菌株基本上聚类在同一组群内。在温室中对供试菌株进行致病性测定,结果表明所有菌株对水稻品种Tetep都有致病性,菌株间致病力差异显著（α＝0．05）,病情指数范围为0．73－18．7,平均感指病指数为5．24。试验分析结果表明水稻纹枯病菌具有丰富的遗传多样性,不同县市的菌株存在很大的遗传分化现象（FST＝0．579）,RAPD遗传聚类群的划分与菌株的地理来源有明显的相关性,但菌株的致病力差异与菌株的来源、遗传聚类组群的划分没有明显的相关性。     

EcbCSL101菌株hrpN基因的克隆、表达及HarpinEcbCSL101蛋白的生物学活性

胡萝卜软腐欧文氏茵甜菜亚种(Erwinia carotovora subsp.betavasculorum)EcbCSL101菌株具有很强胞外酶分泌活性,接种非寄主植物烟草引起过敏反应.Southern blotting结果表明EcbCSL101菌株中含有hrpN基因.PCR扩增含EcbCSL101完整开放阅读框的DNA片段并克隆到表达载体pET28a( )中.核苷酸序列分析表明,EcbCSL101菌株的hrpN基因的ORF为1113 bp,编码36.65 kD HarpinEcbCSL101蛋白(GenBank,DQ355519),与其它几种软腐欧文氏菌Harpin蛋白有较高的同源性.将含有hrpNEcbCSL101基因的重组质粒转化到大肠杆菌JM109(DE3)中进行表达,纯化后的HarpinEcbCSL101能诱导烟草发生过敏反应.     

真养产碱杆菌聚羟基烷酸合成酶基因在欧文氏菌中的表达

将含有真养产碱杆菌(Alcaligenes eutrophus)合成聚羟基烷酸(PHA)基因(phaCA3)的质粒pTZl8U—PHB改造成为具有卡那霉素抗性的质粒pJMCl．并以电击法将pJMCl引入利用碳源广泛的胡罗卜软腐欧文氏茵(Erwinia carotovora)非致病菌系(Eccl3B)中,phaCAB可获得高效表达,膨大的转基因菌[Eccl3B(pJMCl)]细胞内几乎充满PHA颗粒。以蔗糖为碳源,初步在5L发酵罐中对转基因菌进行分批补料培养35h,菌体干重达28g／L,PHA占菌体干重的68％,具有生产潜力。将该菌合成的PHA提取纯化(纯度达99％)后,进行核磁共振分析,发现它只有单一的聚-β-羟基丁酸(PHB)组分。     

Examination of resistance of potato genotypes to Erwinia ssp

Potato can be attacked by several economically important pathogens. From the various diseases, in our experiment we dealt with the bacterial soft rot of potato caused by Erwinia species. In the experiments back cross progenies (BC1, BC2, BC3 and BC4) of Solanum brevidens + Solanum tuberosum somatic hybrids produced by the Potato Research Centre, Keszthely were tested to the infection of E carotovora ssp. atroseptica (Eca), E. carotovora ssp. carotovora (Ecc) and E. chrysanthemi (Echr). All together 11 BC genotypes pre selected from several hundred breeding lines based on their preferred agronomical appearance and virus resistance characters as well as 4 Hungarian potato cultivar (Rioja, Desiree, White lady and Hópehely) as controls were involved into the experiments. Tuber slices from each genotype were artificially infected with bacteria suspension Ecc strain D3, and Echr strain CHR 1492, and Eca strain BN3) and incubated at 27 degrees C with 100% relative air humidity for 48 h before evaluation. Dry matter and starch content of tubers were determined right before the tests. Volume of rotted tuber tissue was determined in mm3 and used for comparison of the level of resistance or susceptibility of the genotypes. Relationship between the reaction to the bacteria strains and dry matter content was examined also. Tested genotypes showed the highest resistance to Eca, while the highest susceptibility to Echr. By the increase of BC level the susceptibility of the genotypes significantly increased as well regardless of the tested bacteria. No direct correlation was found between the dry matter content of tubers and their reaction to tested bacteria.     

Enumeration of two competing Erwinia carotovora populations in potato tubers by a membrane filter-immunofluorescence procedure

Relative increases in cell populations of specific Erwinia carotovora strains injected into potato tubers either singly or in combination of two strains were determined by depositing tissue extracts on membrane filters and staining cells of individual strains by immunofluorescence. The population increase of an Erw. carotovora subsp. atroseptica (Eca) strain was, in general, not affected by the presence of an Erw. carotovora subsp. carotovora (Ecc) strain. However, the increase of an Ecc strain was inhibited by the presence of an Eca strain, especially at incubation temperatures of 5° and 15°C but not at 26°C. One Ecc strain consistently increased in population over another Ecc strain at a greater rate when they were inoculated together at the same loci in comparison to when they were inoculated separately at different loci.     

Use of TnphoA to enrich for extracellular enzyme mutants of Erwinia carotovora subspecies carotovora

Soft rot Erwinia species secrete a range of enzymes into the extracellular environment. Therefore, the genetically amenable Erwinia system is a useful model for the study of protein secretion by Gram-negative bacteria. We have used a lambda-sensitive derivative of Erwinia carotovora subspecies carotovora (Ecc) and the transposon TnphoA, to isolate a range of extracellular enzyme mutants. The use of TnphoA provides an enrichment for extracellular enzyme mutants over other transposon-based systems. In these mutants, the alkaline phosphatase activity of the hybrid protein is found in the periplasm, and is under the control of the Ecc promoters. Three TnphoA-induced extracellular enzyme mutants were studied in detail. One proved to be an enzyme structural gene mutant, whilst the other two appeared to be secretory mutants.     

Erwinia carotovora ssp.carotovora Infection Induced“Defense Lignin”Accumulation and Lignin Biosynthetic Gene Expression in Chinese Cabbage(Brassica rapa L.ssp.pekinensis)

Erwinia carotovora subsp. carotovora (Ecc) infects and causes soft rot disease in hundreds of crop species includingvegetables, flowers and fruits. Lignin biosynthesis has been implicated in defensive reactions to injury and pathogeninfection in plants. In this work, variations of lignin content and gene expression in the molecular interaction betweenChinese cabbage and Ecc were investigated. H_2O_2 accumulation and peroxidase activity were detected by 3, 3-Dimethoxybenzidine staining at mocked and Ecc-inoculated sites of Chinese cabbage leafstalks. Mason lignin contentin inoculated plants increased by about 7.84%, 40.37%, and 43.13% more than that of the mocked site at 12, 24 and 72 hafter inoculation, respectively. Gas chromatography detected more p-coumaryl (H) and less coniferyl (G) and sinapyl (S)monolignins in leafstalks of Chinese cabbage. All three monomers increased in Ecc-infected leafstalks, and the Ecc-induced"defense lignin" were composed of more G and H monolignins, and less S monolignin. After searching the expressedsequence tags (EST) data of Chinese cabbage, 12 genes putatively encoding enzymes involved in lignin biosynthesis wereselected to study their expression. All of these genes could be induced by mock inoculation and Ecc infection, while thegene expression lasted for several more hours in the infected samples than in mocked and untreated plants. Our resultsindicated that "defense lignin" was different from the developmental lignin in composition; G and S monolignins weresignificantly induced in plants in response to the soft rot Ecc; thus, lignin biosynthesis was differentially regulated andplayed a role in plant response to the soft rot Ecc.     

Haemagglutinins and fimbriae of soft rot Erwinias.

Strains of phytopathogenic soft rot Erwinia spp. were examined for haemagglutinin (HA) production. Mannose-sensitive HA was found only in five of 15 strains of E. carotovora subsp. carotovora. Mannose-resistant HA (MRHA) was found in 12 of 15 strains of E.c. carotovora, ten of 13 strains of E.c. subsp. atroseptica and the single strain of E.c. subsp. betavasculorum, as well as all seven strains of E. chrysanthemi. MRHA, detectable only in a microtitre tray HA assay was of either broad- or narrow-spectrum activity when examined against blood of seven different animal species and could be inhibited by the beta-galactoside asialofetuin. Fimbriae of ca 10 nm diameter were found on MRHA(+) bacteria E.c. carotovora and E.c. atroseptica.     

Differentiation of Erwinia carotovora subsp. carotovora and Erwinia carotovora subsp. atroseptica isolated from potato by Western blot and subsequent indirect ELISA

Electrotransfer of protein bands from a polyacrylamide gel to a hydrophobic poly-vinylidene difluoride (PVDF) membrane (Western blot) and their serological determination by indirect ELISA (immunoblotting) were used to differentiate Erwinia carotovora subsp. carotovora (Ecc) from Erwinia carotovora subsp. atroseptica (Eca). Ninety strains: 69 Ecc, 19 Eca and two Erwinia chrysanthemi (Echr) were examined. Eight polyclonal antisera against whole cells, glutaraldehyde fixed cells, glycopro-teins, and somatic antigens were prepared. Antisera produced with glutaraldehyde fixed cells did not recognize any band of the protein pattern. The remaining antisera recognized a limited number of bands. Two protein bands allowed differentiation of the two subspecies by the antisera against glycoproteins. One band with an estimated molecular weight of 36000 Da was present in the 19 Eca strains tested and another band with an estimated molecular weight of 35 000 Da was present in the 69 Ecc strains, except for three cases. The strains of Echr showed a band with an estimated weight of 33 000 Da.     

Serological characterization of potato isolates of Erwinia carotovora subsp. atroseptica and subsp. carotovora using polyclonal and monoclonal antibodies

Serological, biochemical and physiological characteristics of 81 strains of Erwinia carotovora subsp. atroseptica ( Eca ) and 67 strains of subsp. carotovora ( Ecc ) from potato, isolated in Spain and from several international collections, have been studied. Ouchterlony double diffusion (ODD), indirect immunofluorescence (IIF) and indirect enzyme-linked immunosorbent assay (ELISA) were the methods used. The antibodies were polyclonals from eight antisera prepared with Eca serogroup I and Ecc serogroup III and two monoclonal antibodies (MAbs), 4G4 from Spain and 4F6 from Canada, both prepared with Eca strains of serogroup I. Serogroup I for Eca and several serogroups for Ecc were the most commonly found in the collection studied. Serological relationships between Eca and Ecc independently of the serogroups were observed by IIF and ELISA using polyclonal antibodies. Common epitopes between all Eca and Ecc studied were detected. Both MAbs recognized epitopes in Eca strains of serogroups I and XXII in IIF and ELISA but they did not react with strains of other serogroups nor Ecc strains. The pattern of reaction against the strains assayed was rather similar but not identical indicating that they represent two different and well conserved epitopes. This study confirms the serological complexity of Ecc and Eca and gives information about the serological probes for detection of both subspecies.     

Haemagglutinins and fimbriae of soft rot Erwinias

A. WALLACE AND M.C.M. PÉROMBELON. 1992. Strains of phytopathogenic soft rot Erwinia spp. were examined for haemagglutinin (HA) production. Mannose-sensitive HA was found only in five of 15 strains of E. carotovora subsp. carotovora. Mannose-resistant HA (MRHA) was found in 12 of 15 strains of E.c. carotovora, ten of 13 strains of E.c. subsp. atroseptica and the single strain of E.c. subsp. betavasculorum, as well as all seven strains of E. chrysanthemi. MRHA, detectable only in a microtitre tray HA assay was of either broad- or narrow-spectrum activity when examined against blood of seven different animal species and could be inhibited by the β-galactoside asialofetuin. Fimbriae of ca 10 nm diameter were found on MRHA⁺ bacteria of E.c. carotovora and E.c. atroseptica.     

Specificity of an antiserum against Erwinia carotovora subsp. atroseptica in indirect ELISA

Attempts to differentiate Erwinia carotovora subsp. atroseptica (Eca) from Erwinia carotovora subsp. carotovora (Ecc) by indirect ELISA using polyclonal antisera against the former bacterium were unsuccessful. However, when bacterial cells were preincubated with an antiserum against Eca serogroup I and excess serum washed away prior to coating on micro-ELISA plates, specificity was improved. This modified indirect ELISA was able to separate Eca serogroups I, XVIII and XXII from all the Ecc serogroups tested. Cross adsorption of the antiserum with Ecc serogroup XXIX resulted in greatly reduced absorbance values for all strains/serogroups except Eca serogroups I and XXII. Cross adsorption with the homologous Eca strain reduced absorbance values for all strains/serogroups. It is suggested that the differentiation of Eca serogroups I and XXII obtained with the modified indirect ELISA could be attributed to the removal of antibodies cross reacting to soluble antigens and the retention of antibodies to specific cell surface antigens.