丁香假单胞菌发酵液中的冠菌素含量测定方法及其应用

为了辅助提高工程菌种改良的效率,建立了一种简单、快速、高效的分散液-液微萃取-高效液相色谱检测丁香假单胞菌发酵液中冠菌素的分析方法。优化了蛋白沉淀法、萃取剂的种类和体积、分散剂的种类和体积、萃取时间和离心强度等对萃取率的影响。确定最优萃取条件为:以2.0 mL丙酮作为分散剂,以400 μL氯苯为萃取剂,萃取5 min,在5 000 r/min下离心5 min。高效液相色谱的检测条件为:流动相为V (甲醇) : V (0.5%乙酸水溶液) = 70 : 30,等梯度洗脱,流速1.0 mL/min,柱温30 ℃,检测波长220 nm。在3.2~100 mg/L范围内,冠菌素的峰面积与其质量浓度间呈良好的线性关系,相关系数 (r) 为0.999 8。方法的检出限为2.1 mg/L,定量限为6.5 mg/L。在6.5、25和100 mg/L添加水平下,冠菌素在丁香假单胞菌发酵液中的回收率在95%~98%之间,相对标准偏差 (n = 5) 在2.7%~5.1%之间。该方法可用于丁香假单胞菌发酵液中冠菌素含量的测定。     

构树上一种新的丁香假单胞菌致病变种的研究

 构树细菌性疫病是一种荧光假单胞菌引起的新病害。主要症状有叶片角斑,嫩梢肿大和幼枝溃疡。从江苏一带分离获得的12个构树菌株和3个桑树对比菌株进行交互接种试验,发现构树菌株与桑树菌株之间不能交互侵染其寄主。细菌学特征和LOPAT试验及其它37项生理生化和营养特性试验表明,两种菌的表型特征基本相似,仅在7种化合物的利用上存在差异。两种菌的血清学反应无相关性。细胞全蛋白SDS一聚丙烯酰胺凝胶电泳图谱也略有不同。试验结果证明,构树细菌性疫病细菌属于假单胞菌属(Pseudomonas),丁香假单胞菌(P.syringae)的一个新致病变种,定名为Pseudomonas syrzngae pv.broussonetiae pv.nov.     

丁香假单胞菌对亚洲玉米螟幼虫抗寒能力的影响

本文用丁香假单胞菌Pseudomonas syringae处理亚洲玉米螟Ostrinia furnacalis(Guenée)幼虫,使用过冷却仪测定过冷却点,分析该菌株对参试幼虫耐寒能力的影响。结果表明,亚洲玉米螟5龄非滞育幼虫抗寒能力比其他龄期非滞育幼虫强。亚洲玉米螟滞育5龄幼虫抗寒能力比非滞育5龄幼虫强。使用丁香假单胞菌处理亚洲玉米螟5龄非滞育和滞育幼虫,24 h后非滞育幼虫的过冷却点和结冰点分别提高了5.14和2.83℃。5龄滞育幼虫过冷却点和结冰点分别提高了11.25和4.65℃。丁香假单胞菌菌悬液(浓度为8×108 cfu/m L)对亚洲玉米螟具有较好的杀虫效果,非滞育5龄幼虫在(15±1)℃条件下处理24 h后平均死亡率39.1%。     

冠菌素对玉米抗倒伏能力及产量的影响

在田间条件下,以玉米品种先玉335和郑单958为材料,在八展叶时于叶面喷施不同浓度 (0、0.01、0.1、1和10 μmol/L) 冠菌素 (COR),研究了COR对玉米抗倒伏能力及产量的影响。结果表明:经10 μmol/L COR处理后,先玉335的株高和穗位高分别比对照降低了7.0%和19.9%,郑单958分别降低了20.8%和18.2%,单株叶面积、节间抗折断力、穗下第8~14节间长度和节间最大直径与对照间亦差异显著。且随着COR浓度的增加,先玉335和郑单958的株高、穗位高、单株叶面积和穗下第8~14 节间长度均逐渐降低;第8~14节间最大直径和节间抗折断力逐渐增大。经1 μmol/L COR处理后,2个供试玉米品种的产量、穗数、穗粒数和千粒重较对照均有所增加,其中先玉335的产量较对照增加了9.9%,郑单958增加了4.3%。综上所述,COR可以提高玉米的抗倒伏能力,不同品种玉米对COR的敏感性不同,其中先玉335抗倒伏的最适COR浓度为10 μmol/L,郑单958抗倒伏的最适COR浓度为1 μmol/L。     

荧光假单胞菌不同施用方式对田间烟草青枯病发生的影响

烟草青枯病在我国西南烟区普遍发生流行,是影响烟叶品质和产量的主要病害.为减轻烟草青枯病的危害,本研究以烤烟主栽品种"云烟87"为试验对象,通过田间小区试验探究荧光假单胞菌的不同施用方式对烟草青枯病发生以及对烟草生长发育的影响.结果表明,移栽期窝施5 g菌肥、100 mL微生物菌液灌根以及移栽期窝施5 g菌肥且灌根微生物...     

荧光假单胞菌2P24中phlF基因对抗生素2,4-二乙酰基间苯三酚产生的影响

Pseudomonas fluorescens2P24是分离自山东小麦全蚀病自然衰退土的1株生物防治菌株,产生抗生素2,4-二乙酰基间苯三酚(2,4-diacetylphloroglucinol;2,4-DAPG)是其主要防病机制。2,4-DAPG是由phlACBD基因簇合成,受多种调控因子调控。本研究用Tn5转座子插入技术,获得1株phlA基因转录增强的突变体,其突变基因为抗生素合成的负调控基因phlF。与野生菌相比,phlF基因的缺失突变体中phlA的转录增强约100倍,抗生素产量提高492倍。同时,菌株2P24的phlF缺失突变体对病原真菌的拮抗作用明显增强。但2,4-DAPG过量表达菌株对多种作物种子根生长有抑制作用。     

拮抗青枯劳尔氏菌的荧光假单胞菌SN15-2分离鉴定及其生防能力分析

为了筛选出对番茄青枯病具有较好防效的生防菌,采用皿内测定法从上海地区的番茄青枯病自然衰退土壤中,分离到一株对番茄青枯病有很强抑制作用的菌株SN15-2,并进行了分子鉴定和对荧光假单胞菌SN15-2产抗生素能力和定殖能力测定。结果表明,菌株SN15-2为荧光假单胞菌(Pseudomonas fluorescens)。其菌株能产生2,4-二乙酰基间苯三酚(2,4-DAPG)、硝吡咯菌素(PRN)、藤黄绿脓菌素(PLT)。同时,可以产生HCN、噬铁素,能够形成生物膜。SN15-2在施入番茄根际后的前20 d定殖数量减少,20 d后基本稳定,60 d时,在干土中的定殖数量可达3.67×105cfu/g。盆栽防效分析表明,荧光假单胞菌SN15-2对番茄青枯病防效达到46.58%。     

烟草根际细菌铜绿假单胞菌swu31-2的定殖能力及其对烟草青枯病的防治作用

从重庆黔江烟草田间分离获得一株烟草青枯菌拮抗菌株Pseudomonas aeruginosa swu31 2（简称swu31 2）。采用逐步提高药物浓度的方法,筛选获得了抗链霉素300 μg/mL对烟草青枯病菌拮抗活性稳定的swu31 2突变菌株。采用灌根接种法,研究其在烟草根、茎和叶表面及内部的定殖能力及其对烟草青枯病的防治作用。结果表明,swu31 2能在烟草各组织的表面及内部定殖。该菌株在烟草各组织内部的数量均表现为“由增到减”的趋势。在接种后第8天定殖数量达到最高峰,随后有所下降;到第20天各组织内的数量仍然维持较高水平（105 cfu/g 以上）。同时,在接种20 d后,烟草的根、茎和叶的表面仍然可以检测到swu31 2的存在。盆栽试验结果表明,swu31 2的菌液和活性物质对烟草青枯病均有一定的防治效果。其中先施swu31 2菌液和活性物质粗提物的防效好于农用链霉素（51.25%）,分别为60.87%和 60.32%,而后施菌液和活性物质粗提物的防效也分别达39.50%和20.90%。     

生防荧光假单胞菌CPF-10对西瓜根围土壤真菌群落的影响

探讨了生防菌荧光假单胞菌Pseudomonas fluorescens CPF-10对西瓜根围土壤真菌群落结构的影响。采用传统分离培养法,结合变性梯度凝胶电泳(DGGE)分析,研究了施用CPF-10后不同生育期西瓜根围土壤真菌群落结构的变化。结果表明:施入生防菌CPF-10后2周,其对土壤真菌有一定的促进作用;第3～7周时,CPF-10对土壤真菌尤其是部分病原菌有较强的抑制作用;CPF-10对丛枝菌根真菌(AMF)有一定的促进作用,可维持3周左右;收获期后则检测不到CPF-10对土壤真菌群落的影响。生防菌CPF-10对西瓜根围土壤真菌群落产生的短暂影响不会对土壤生态系统构成长期威胁。对比土著真菌及丛枝菌根真菌的DGGE图谱和切胶条带测序结果,发现DGGE技术更适用于分析小范围特定菌属如丛枝菌根真菌的变化。     

内生菌荧光假单胞菌DLJ1和蜡状芽胞杆菌SZ5对南方根结线虫胁迫下辣椒植株抗性与产量品质的影响

为了检验内生菌荧光假单胞菌DLJ1和蜡状芽胞杆菌SZ5的促生与提高植株对南方根结线虫的防治效果, 确定合适的菌剂接种方式, 并分析其机制, 本文以杀线剂阿维菌素为对照, 比较了不同接菌方式下DLJ1 和SZ5对辣椒植株在南方根结线虫胁迫下的抗性以及对产量品质的影响?结果证实DLJ1在种子萌发期的一次接菌与萌发期与田间的5次接菌处理均可使根系根结数减少70%以上, 使植株对南方根结线虫的抗性升至高抗级别, 辣椒产量提高超过150%; DLJ1一次接菌处理组的果型最大?Vc与可溶性蛋白含量最高?SZ5萌发期一次处理根结数降低40%, 植株对南方根结线虫表现为中抗, 产量提高60%以上; SZ5多次接菌处理根结数只能降低27%, 产量仅提高6%左右?阿维菌素处理的效果介于DLJ1和SZ5之间, 可降低植株根结数45%, 提高产量49%左右?分析其机理发现, 两菌剂处理均可通过提高植株的POD酶活性和总酚含量, 降低O2-产生速率和MDA积累, 提高光合色素含量等来促进植物生长, 通过提高几丁质酶活性来提高植株对线虫的抗性?总之, 只需要在辣椒种子萌发期进行一次DLJ1接菌处理, 即可使植株获得稳定而显著的促生和防治南方根结线虫双重效果, 提示DLJ1菌在农业生产上的应用前景广阔?     

实时荧光定量PCR法检测十字花科细菌性黑斑病菌

为有效防控我国的检疫性有害生物十字花科细菌性黑斑病菌Pseudomonas syringae pv.maculicola在国内的传播与蔓延,通过设计1对特异性引物3539,利用132株靶标和非靶标菌为模板进行PCR扩增,建立了实时荧光定量PCR法,并进行了模拟种子带菌试验。结果显示,引物3539为只针对十字花科细菌性黑斑病菌扩增出的特异性产物;在模拟种子带菌检测中,常规PCR对菌悬液的检测限为10~5CFU/m L,实时荧光定量PCR的检测限为10~3CFU/m L,其中10~8CFU/m L菌液的Ct值最低,为22.90,10~3CFU/m L菌液的Ct值最高,为35.73,且不同浓度菌液间的Ct值均有显著差异;不同带菌率模拟种子的检测结果表明,常规PCR和实时荧光定量PCR能检测到的带菌率分别为0.5%和0.1%。研究表明,实时荧光定量PCR法不仅可用于病种的检测,也可用于病害的早期诊断。     

Polyphosphate kinase is essential for swarming motility, tolerance to environmental stresses, and virulence in Pseudomonas syringae pv. tabaci 6605

Polyphosphate kinase (PPK), encoded by the ppk gene, is a principal enzyme responsible for synthesis of inorganic polyphosphate (poly P) from ATP in many Gram-negative bacteria. In order to elucidate the functions of poly P in Pseudomonas syringae pv. tabaci 6605, an in-frame deletion mutant of the ppk gene (ppk) was constructed. The ppk mutant did not accumulate poly P, whereas the wild-type strain accumulated a large quantity. The mutant had reduced swarming motility, even though it retains swimming motility like the parental strain. The mutant exhibited increased sensitivity to prolonged incubation and environmental stresses, such as heat shock and oxidative stress and reduced exopolysaccharide (EPS) production compared to the wild-type. Northern blot analysis revealed that expression of the rpoS gene, encoding the stationary phase sigma factor RpoS, was reduced in ppk in the logarithmic phase, indicating that rpoS is regulated by the ppk gene. The poly P deficient mutant had significantly reduced ability to cause disease in its host tobacco plant and in planta growth of the mutant was also significantly reduced in host tobacco leaves as compared to the wild-type strain. Thus, our results suggest that poly P plays an important role in the virulence of P. syringae pv. tabaci 6605.     

进境加拿大豌豆中豌豆细菌性疫病菌的检测

 利用MT选择性培养基从进境加拿大豌豆样品上分离到一株细菌分离物(编号1314),对该分离物进行PCR检测、16S和23S rRNA序列扩增、多位点序列分析、Biolog测定、烟草过敏性反应和致病性测试。豌豆细菌性疫病菌(Pseudomonas syringae pv. pisi, Ppi)特异引物AN7F/AN7R扩增分离物1314和Ppi菌株ATCC 11043得到预期272 bp的条带,二者的PCR产物序列一致,与GenBank中Ppi (X97405)的序列相似性为99.57%。分离物1314的部分16S rRNA、23S rRNA序列以及16S-23S序列均与Ppi菌株ATCC 11043一致。选择gap1、gltA、gyrB和ropD 4个看家基因进行多位点序列分析,系统发育树显示分离物1314与Ppi菌株聚在同一组内。Biolog鉴定结果表明:分离物1314为Ppi,PROB值为0.898,SIM为0.72。该分离物人工接种豌豆茎秆能引起水渍状症斑,接种烟草叶片产生过敏性反应。基于上述试验结果,将分离物1314鉴定为豌豆细菌性疫病菌。     

A functional gene-for-gene interaction is required for the production of an oxidative burst in response to infection with avirulent Pseudomonas syringae pv. tomato in Arabidopsis thaliana

An early event correlated with the gene-for-gene hypersensitive response (HR) is the accumulation of active oxygen species (AOS), also known as the oxidative burst. We present data that genetically demonstrates that the oxidative burst is a downstream component of the RPS2- avrRpt2gene-for-gene signal cascade. An in planta AOS assay using the fluorescent probe 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) was modified for use with the Arabidopsis thaliana / Pseudomonas syringae pv.tomato (P. syringae pv. tomato) model system. An oxidative burst occurred between 8 and 15 hpi with avirulent P. syringae pv. tomato(avrRpt2), but not with virulent P. syringae pv. tomato. This burst preceded cell death and was not observed in the RPS2 Arabidopsis mutantsrps2-101C and rps2-201 inoculated with avirulent P. syringae pv. tomato. An HR-like response has been observed when plants undergoing a systemic acquired resistance (SAR) response are challenged with a normally virulent pathogen (manifestation stage of SAR), however an HR-like oxidative burst was not detected by the in planta AOS assay during this stage of SAR.     

Identification and in planta detection of Pseudomonas syringae pv. tomato using PCR amplification of hrpZPst

A rapid detection method based on PCR amplification of Pseudomonas syringae pv. tomato chromosomal sequences was developed. Primer design was based on the P. syringae DC3000 hrpZ_Pst gene, which maps on a pathogenicity-associated operon of the hrp/hrc pathogenicity island.A 532 bp product corresponding to an internal fragment of hrpZ_Pst was amplified from 50 isolates of P. syringae pv. tomato belonging to a geographically representative collection. The amplification product was also obtained from three coronatine-deficient strains of P. syringae pv. tomato.On the other hand, PCR did not produce any such products from 100 pathogenic and symbiotic bacterial strains of the genera Pseudomonas, Xanthomonas, Erwinia, and Rhizobium and 75 unidentified bacterial saprophytes isolated from tomato plants. The method was tested using leaf and fruit spots from naturally-infected tomato plants and asymptomatic nursery plants and artificially contaminated tomato seeds. The results confirmed the high specificity observed using pure cultures.     

Nucleotide Sequence and Organization of Copper Resistance Genes from Pseudomonas syringae pv. actinidiae

Copper-containing bactericides have been used to control bacterial canker of kiwifruit, caused by Pseudomonas syringae pv. actinidiae. However, the efficacy of copper has been reduced by the occurrence of copper-resistant strains. Analysis of the DNA sequence of a cluster region containing the copper-resistance genes from P. syringae pv. actinidiae suggested the presence of three possible different systems for copper resistance: copper-trapping, copper-efflux and copper-transport systems. Transposon insertional inactivation analysis indicated that the copper-trapping system was essential for copper resistance.     

Genetic analyses of Pseudomonas syringae isolates from Belgian fruit orchards reveal genetic variability and isolate-host relationships within the pathovar syringae, and help identify both races of the pathovar morsprunorum

A collection of Pseudomonas syringae and viridiflava isolates was established between 1993 and 2002 from diseased organs sampled from 36 pear, plum and cherry orchards in Belgium. Among the 356 isolates investigated in this study, phytotoxin, siderophore and classical microbiology tests, as well as the genetical methods REP-, ERIC- and BOX- (collectively, rep-) and IS50-PCR, enabled identification to be made of 280 isolates as P. syringae pv. syringae (Pss), 41 isolates as P. syringae pv. morsprunorum (Psm) race 1, 12 isolates as Psm race 2, three isolates as P. viridiflava and 20 isolates as unclassified P. syringae. The rep-PCR methods, particularly BOX-PCR, proved to be useful for identifying the Psm race 1 and Psm race 2 isolates. The latter race was frequent on sour cherry in Belgium. Combined genetic results confirmed homogeneities in the pvs avii, and morsprunorum race 1 and race 2 and high diversity in the pv. syringae. In the pv. syringae, homogeneous genetic groups consistently found on the same hosts (pear, cherry or plum) were observed. Pathogenicity on lilac was sometimes variable among Pss isolates from the same genetic group; also, some Psm race 2 and unclassified P. syringae isolates were pathogenic to lilac. In the BOX analyses, four patterns included 100% of the toxic lipodepsipeptide (TLP)-producing Pss isolates pathogenic to lilac. Many TLP-producing Pss isolates non-pathogenic to lilac and the TLP-non-producing Pss isolates were classified differently. Pseudomonas syringae isolates that differed from known fruit pathogens were observed in pear, sour cherry and plum orchards in Belgium.     

Note: Comparison of three laboratory methods to evaluate the pathogenicity and virulence of tenPseudomonas syringae pv.syringae strains on apple, pear, cherry and peach trees

The pathogenicity and virulence of ten GreekPseudomonas syringae pv.syringae strains from different hosts (citrus, pear, apple, peach and cherry) were evaluated using three different laboratory methods, which produced results in good agreement. All ten strains were virulent on apple, pear, cherry and peach trees. The extent of tissue colonized varied considerably among strains and cultivars. On excised shoots and twigs of apple and pear, strains BPI 176, BPI 203, PI 2 and PI 14 were the most virulent and strains BPI 689, BPI 992, BPI 4, BPI 20, PI 18 and PI 19 were the least virulent. On excised shoots and twigs of peach and cherry, strains BPI 176, BPI 203, PI 2, PI 14, PI 18 and PI 19 were the most virulent and strains BPI 4 and BPI 20 were the least virulent. Moderate virulence was evinced by strains BPI 689 and BPI 992. These pathogenicity assays are proposed as rapid and reproducible screening systems to evaluate the susceptibility of apple, pear, cherry and peach cultivars to this bacterial pathogen.     

在小檗碱作用下水稻细菌性条斑病菌生理及转录反应分析

为探究小檗碱对水稻细菌性条斑病菌Xanthomonas oryzae pv. oryzicola的抑菌机制,测定小檗碱对水稻细菌性条斑病菌GX13菌株的毒力、致病因子、细胞结构和能量代谢的影响,并利用Illumina-HiSeq高通量测序技术分析小檗碱对水稻细菌性条斑病菌菌体基因表达的影响。结果表明:经15μg/mL小檗碱处理后,GX13菌株侵染水稻品种玉丝占后引起水稻细菌性条斑病的病情指数为6.70,低于对照组;每1×108CFU菌体胞外多糖产量为1.58μg,显著低于对照的2.42μg;纤维素酶、蛋白酶、淀粉酶等胞外酶活性均低于对照组;与能量代谢相关的苹果酸脱氢酶(MDH)活性为1.42 U/g prot,低于对照组,而丙酮酸和三磷酸腺苷含量升高,分别为1.71μmol/mL和379.49μmol/g prot,均高于对照组。同时,细菌的生物膜形成受到抑制,细胞结构受到破坏。转录反应分析发现,与II型分泌系统相关的xpsD、xpsF和xpsM等基因、与III型分泌系统相关的hrcC、hrcJ、hrcN和hrcQ等基因、与胞外多糖分泌相关的gumF和gumI基因,以及与细胞结构与功能...     

Suppression of Rhizoctonia root-rot of tomato by Glomus mossae BEG12 and Pseudomonas fluorescens A6RI is associated with their effect on the pathogen growth and on the root morphogenesis

Rhizoctonia solani root-rot is a major soilborne disease causing growth and yield depression. The ability of Glomus mosseae BEG12 and Pseudomonas fluorescens A6RI to suppress this soilborne disease in tomato was assessed by comparing the shoot and root growth of plants infested with R. solani 1556 when protected or not by these beneficial strains. The epiphytic and parasitic growth of the pathogenic R. solani 1556 was compared in the presence and absence of the biocontrol agents by microscopical observations allowing the quantification of roots with hyphae appressed to epidermal cells (epiphytic growth) and of roots with intraradical infection (parasitic growth). The root architecture of the tomato plants under the different experimental conditions was further characterized by measuring total root length, mean root diameter, number of root tips and by calculating degree of root branching. G. mosseae BEG12 and P. fluorescens A6RI fully overcame the growth depression caused by R. solani 1556. This disease suppression was associated with a significant decrease of the epiphytic and parasitic growth of the pathogen together with an increase of root length and of the number of root tips of inoculated tomato plants. The combined effects of G. mosseae BEG12 and P. fluorescens A6RI on pathogen growth and on root morphogenesis are suggested to be involved in the efficient disease suppression.