假茄科雷尔氏菌引起向日葵青枯病在中国的首次报道

茄科雷尔氏菌复合种侵染引起的青枯病是众多作物上的毁灭性病害。2020年笔者首次在广东省东莞市发现向日葵青枯病,并对其病原菌进行了鉴定。室内人工接种试验、16S rDNA序列比对和演化型鉴定结果表明,引起向日葵青枯病的病原菌为假茄科雷尔氏菌Ralstonia pseudosolanacearum。生理生化特性和致病性鉴定结果表明,分离自向日葵的15株假茄科雷尔氏菌为1号生理小种和生化变种3。egl基因部分序列系统进化分析表明,15株假茄科雷尔氏菌分属4个序列变种,其中8株菌株为序列变种17,5株菌株为序列变种13,其余2株菌株分别为序列变种14和序列变种54。本文是我国首次报道假茄科雷尔氏菌侵染引起向日葵青枯病。     

胜红蓟青枯病病原鉴定及其生物学特性

胜红蓟青枯病是我国发生的一种新病害。为了明确引起胜红蓟青枯病的病原,对分离自广东的胜红蓟青枯病菌的菌落形态、16S rDNA序列、碳水化合物利用、致病性及演化型等进行了分析。在含1% 2,3,5-氯化三苯基四氮唑选择性培养基平板上,病原菌的菌落呈近圆形或梭形,隆起,中间粉红色,周围乳白色。应用细菌通用引物27f和1541r扩增16S rDNA,Ac-YcSj-11-1~Ac-YcSj-11-10 10个菌株的16S rDNA近全长均为1 421 bp,且与茄科雷尔氏菌Ralstonia solanacearum GMI1000 16S rDNA的同源率为100%。该病原菌可以利用麦芽糖、乳糖、纤维二糖、山梨醇、甘露醇和卫矛醇等6种碳水化合物,并可侵染姜、沙姜、番茄,弱侵染茄子、辣椒。10个菌株均可同时扩增得到144 bp的演化型Ⅰ特异带和280 bp的茄科雷尔氏菌特异带。研究表明,引起广东胜红蓟青枯病的病原菌为茄科雷尔氏菌R.solanacearum,且属于4号生理小种、生化变种Ⅲ和演化型Ⅰ（亚洲组）。     

洋葱伯克霍尔德氏菌JX-1防治番茄青枯病机理的初步分析

本研究分离得到一株对茄科雷尔氏菌Ralstonia solanacearum具有明显拮抗作用的根围细菌JX-1，其温室防治番茄青枯病的防效达80.89%。形态学、生理生化、16S rDNA序列和gyrB序列分析表明，该菌株属于洋葱伯克霍尔德氏菌Burkholderia cepacia。生防相关性状分析表明，菌株JX-1可产生嗜铁素、蛋白酶等抗菌物质。抗生素合成相关基因分析发现，菌株JX-1还可能产生硝吡咯菌素和藤黄绿脓菌素。转座子随机突变获得2株明显影响菌株JX-1拮抗能力的突变菌株，其中突变体M1710对茄科雷尔氏菌拮抗作用完全消失，序列分析表明Tn5破坏了与非核糖体寡肽产生相关基因tlpks/nrps；而突变体M645则对茄科雷尔氏菌的拮抗作用显著增强，序列分析表明Tn5破坏了gntR基因。上述结果表明，菌株JX-1可产生多种次生代谢产物，并且tlpks/nrps基因和gntR基因在防治番茄青枯病过程中起到重要调整作用。     

空心菜青枯病病原菌的鉴定

 空心菜青枯病是近年来在广东空心菜产区新发生的病害,病株率一般为5%~20%,严重达50%以上。症状初始表现为植株上部1~2片叶萎蔫、下垂,病叶失去光泽呈灰绿色;随着病情的发展,病叶不断增多,最终整株萎蔫,甚至倒伏。经细菌学鉴定、室内致病性测定、16S rDNA序列比较及系统进化分析,结果表明引起该病害的病原是茄科雷尔氏菌(Ralstonia solanacearum);致病性和碳水化合物利用试验结果表明,该病原菌属茄科雷尔氏菌1号生理小种,生化变种4。     

星油藤青枯病病原菌鉴定

 星油藤(Plukenetia volubilis L.) 是一种重要的藤本油料植物,在我国华南地区广泛种植。青枯病是近两年在海南星油藤种植区发生的新病害,为探究星油藤青枯病菌的基本特性及种下分化情况,本研究对分离的6株代表菌株进行了相关分析。细菌学鉴定及致病性测定结果表明,该病害是由类茄科雷尔氏菌(Ralstonia pseudosolanacearum)侵染引起。同时,从传统分类及分子生物学不同层面分析了星油藤青枯病菌的遗传分化情况。生理小种及生化变种的测试结果表明,星油藤青枯病菌属于1号生理小种和生化变种Ⅲ;16S rDNA和egl基因部分序列聚类分析显示,星油藤青枯病菌属类茄科雷尔氏菌演化型Ⅰ即亚洲分支菌株,序列变种34。     

星油藤青枯病病原菌鉴定

 星油藤(Plukenetia volubilis L.) 是一种重要的藤本油料植物,在我国华南地区广泛种植。青枯病是近两年在海南星油藤种植区发生的新病害,为探究星油藤青枯病菌的基本特性及种下分化情况,本研究对分离的6株代表菌株进行了相关分析。细菌学鉴定及致病性测定结果表明,该病害是由类茄科雷尔氏菌(Ralstonia pseudosolanacearum)侵染引起。同时,从传统分类及分子生物学不同层面分析了星油藤青枯病菌的遗传分化情况。生理小种及生化变种的测试结果表明,星油藤青枯病菌属于1号生理小种和生化变种Ⅲ;16S rDNA和egl基因部分序列聚类分析显示,星油藤青枯病菌属类茄科雷尔氏菌演化型Ⅰ即亚洲分支菌株,序列变种34。     

广东广藿香青枯病病原菌鉴定

 广藿香青枯病是近几年在广东广藿香种植区发生的新病害,细菌学鉴定及致病性测定结果表明,该病害是由茄科雷尔氏菌侵染引起的,且属于1号生理小种和生化变种Ⅲ;分子生物学分析结果进一步显示,广藿香青枯病菌属茄科雷尔氏菌演化型Ⅰ即亚洲分支菌株、序列变种44或序列变种17。     

湖南猕猴桃溃疡病菌16S rDNA和16S-23S rDNA间隔区序列的克隆与分析

为明确湖南省猕猴桃溃疡病其致病菌的种类与特征,以猕猴桃主栽品种红阳、米良1号的溃疡病感病枝条为材料,采用BPA培养基、平板划线和梯度稀释法分离病原菌。利用引物27F/1492R和Psa F1/Psa R2对湖南省猕猴桃溃疡病菌16S r DNA和16S-23S r DNA间隔区序列(ITS)进行PCR扩增并进行核苷酸序列测定及相似性分析。获得Psa-JSHY株系、Psa-LXHY株系以及Psa-LXML株系的16S r DNA基因片段1 383 bp及16S-23S r DNA间隔区序列280 bp,且序列一致。经序列相似性比较表明:所分离株系的16S r DNA与法国181、新西兰ABAC9、意大利IKB4等株系的16S r DNA基因序列一致,与日本的KW11等株系存在3个碱基的差异,相似性为99.78%,与国内分离的11-830-1株系存在4个碱基的差异,相似性为99.71%;16S-23S r DNA-ITS序列与中国SXHY11-1、西班牙EFA131.1、葡萄牙PA838、韩国KBE9等株系的ITS序列一致。构建16S r DNA及16S-23S r DNA-ITS序列的进化树,可以看出所分离的株系与已报道的P.syringae pv.actinidiae各菌株聚在同一个进化枝上。以上结果表明,湖南地区分离的3个猕猴桃溃疡病菌致病株系均属于P.syringae pv.actinidiae。     

一株烟草青枯病生防真菌的筛选与鉴定

从烟田健康土壤中分离得到的59株真菌中,筛选出1株对青枯雷尔氏菌(Ralstonia solanacearum)有较好颉颃效果的真菌,抑菌圈直径为9.5 mm.对该菌株进行了脂肪酸组成分析和形态学观察,并经rDNA ITS区测序,该株真菌鉴定为浅黄新萨托菌(Neosartorya aureola).     

甘肃马铃薯疮痂病病原初步鉴定

对甘肃不同地区马铃薯疮痂病病原进行了分离鉴定.采用盆栽方法进行致病性测定、形态特征、生理生化特性测定和16S rDNA序列分析.结果表明:共有6株菌能使马铃薯块茎出现疮痂症,分别为GP-1、GP-2、GH-1、GH-2、JB-1和JB-2.经鉴定菌株GP-1、JB-2的形态特征、生理生化特性及16S rDNA序列与Streptomyces scabies 87 22菌株均一致;GP-2、JB-1的16S rDNA序列与S.scabies菌株相似性为99％.菌株GH-2和GH-1与S.griseus的16S rDNA序列相似性分别为100％和99％,形态特征一致,但不能以棉籽糖和肌醇为单一碳源,暂定为S griseus.     

生防细菌L5生防相关因子的初步分析及其种类鉴定

从扬州郊区土壤中分离到细菌菌株L5,该菌株抑菌谱广,对多种病原真菌都有明显的拮抗能力,离体叶片生物测定结果表明菌株L5对番茄灰霉病防效达72%.对该菌株的生防相关性状进行分析,结果表明菌株L5可产生硝吡咯菌素(Pyrrolnitrin)和嗜铁素等抗菌物质.经过16S rDNA序列、16S-23S rDNA间区序列同源性分析和生理生化性状的测定,将该菌株鉴定为气味沙雷氏菌(Serratia odorifera).细菌菌株L5包含2个16S-23S rDNA的间区(IGSs),其中IGS1内含tRNA Glu基因,间区类型属于IGSG;IGS2内含tRNA lle和tRNA Aia基因,间区类型属于IGS LA.     

Determination of Ralstonia (Pseudomonas) solanacearum rDNA subgroups by PCR tests

Rapid and sensitive polymerase chain reaction (PCR) methods are described for determination of the two 16 S rDNA subgroups of Ralstonia solanacearum, the causal agent of bacterial wilt. A third subgroup consisting of Indonesian R. solanacearum isolates belonging to Division II, the blood disease bacterium and Pseudomonas syzygii can also be identified. Primers were designed to sequences within R. solanacearum 16 S rDNA (equivalent to Escherichia coli 16 S rDNA positions 74–97, 455–475, 1454–1474), and the internal transcribed spacer region between the 16 S and 23 S rDNA genes. Different combinations of forward and reverse primers allowed selective PCR amplification of (a) R. solanacearum Division I (biovars 3, 4 and 5), (b) Division II (biovars 1, N2, and 2) including the blood disease bacterium and P. syzygii , or (c) amplification of Division II only except for five biovar 1, 2 or N2 isolates of R. solanacearum from Indonesia, P. syzygii and the BDB. A total of 104 R. solanacearum , 14 blood disease bacterium and 10 P. syzygii isolates were tested. Simultaneous detection of species and subdivision was achieved by designing a multiplex PCR test in which a 288-base pair (bp) band is produced by all R. solanacearum isolates, and an additional 409-bp band in Division I strains.     

Strain Level Identification of Pseudomonads Using Denaturing Gradient Gel Electrophoresis of 16S-23S Spacer Region rDNA

Specific Pseudomonas strains were detected by PCR amplification of the 16S-23S rDNA spacer region followed by denaturing gradient gel electrophoresis (DGGE) to generate DNA banding profiles. In initial studies, two diverse sequence areas within the 16S-23S rDNA spacer region were located in five closely related Pseudomonas fluorescens and P. putida strains. DNA banding profiles of 16 different pseudomonads were generated using PCR primers flanking this region, followed by DGGE of the PCR products. Distinct banding profiles were observed for each strain, and specificity could be increased by designing additional primers within the spacer region. A specific primer (513-1) was used to selectively amplify and detect a plant-disease suppressive bacterium, P. fluorescens strain 513, in soil. Six field soils from different locations were used with the 513-1 primer to test the specificity of this technique. Five soils did not yield any gel bands, but one soil led to a faint 250-bp band, similar in size to that of P. fluorescens 513. Resolution of strain 513 from the indigenous strain in this soil was achieved by DGGE of the amplified DNA fragments. The results therefore demonstrate the utility of PCR-DGGE analysis for strain-specific identification of pseudomonads in environmental samples. Received 17 January 2000/ Accepted in revised form 10 May 2000     

洛阳核桃黑斑病病原菌鉴定

<正>0引言核桃黑斑病是核桃的三大主要病害之一,发病范围广,危害严重,一般植株受害率70%～90%,果实受害率10%～40%,严重时达65%以上,导致核桃栽培经济效益大幅降低^[1]。核桃黑斑病病菌在病枝、芽鳞和残留的病果等处越冬,从气孔、皮孔、柱头、伤口等处侵入,借雨水、花粉、昆虫传播,可多次侵染^[2]。     

云南花生丛枝植原体16S rRNA和核糖体蛋白基因序列分析

 利用植原体16S rRNA基因及核糖体蛋白基因(ribosomal protein, rp)通用引物对发生在云南元谋的花生丛枝病病株DNA进行PCR扩增,并对扩增片段进行序列测定。扩增获得的云南元谋花生丛枝植原体(PnWB-YNym)16S rDNA、16S-23S rDNA和23S DNA片段总长1 806 bp,rp基因扩增片段长1 171 bp。云南株系与来源于台湾和海南的花生丛枝植原体均有较高同源性。比较16S rDNA片段,发现云南株系在5个位点上与来自台湾或海南的株系存在碱基差异,其中有1个位点的差异是云南元谋株系特异的;再分别比较核糖体蛋白rplV-rpsC 2个基因所编码的氨基酸序列,发现云南株系rpsC编码的第194位氨基酸与台湾和海南的株系存在差异。经16S rDNA片段系统进化及iPhyClassifier在线分析,表明PnWB-YNym在分类上属于16SrII-A亚组成员,与候选种‘Candidatus Phytoplasma australasiae’相关;基于rp基因构建的系统进化树表明,PnWB-YNym与16SrII-A亚组各成员聚为同一亚进化支(iii)。     

生防菌株2P24与CPF-10的鉴定及其生防相关性状的初步分析

 从山东省小麦根围土壤中分离到具有生防活性的细菌菌株2P24、CPF-10。通过对其16S rDNA序列同源性分析及生理生化测定鉴定为荧光假单胞菌(Pseudomonas fluorescens),其中菌株2P24为生物型I,CPF-10为生物型V。对这2株细菌的生防相关性状分析表明,二者均可产生多种抗菌物质,如2,4-二乙酰基间苯三酚(2,4-DAPG)、氢氰酸(HCN)、嗜铁素、蛋白酶等。平板对峙测定表明2株细菌对番茄青枯菌(Rastonia solanacearum)、棉花立枯丝核菌(Rhizoctonia solani)、棉花枯萎菌(Fusarium oxysporum)具有明显的拮抗作用。温室生测表明2P24对番茄青枯病的防效达63.0%。CPF-10达62.4%,且持续稳定。     

Design of Division Specific Primers of Ralstonia solanacearum and Application to the Identification of European Isolates

A PCR diagnostic test for detection of Ralstonia solanacearum at the infraspecific level was developed, based on polymorphisms within the 16S rRNA gene sequence. Partial sequences of this gene were determined for three French isolates which showed the characteristics of R. solanacearum subdivision 2a described by Taghavi et al. (1996). Oligonucleotide primers were designed to incorporate the nucleotide triplet (at positions 458–460 of the 16S rRNA gene) which differs between divisions 1 and 2 16S rRNA sequences of R. solanacearum isolates. A simple PCR test unambiguously revealed the division of each isolate. The PCR test was useful for identification of isolates of R. solanacearum from Europe.     

花生青枯病内生拮抗细菌的鉴定、抗菌活性及其田间防效

从病区健康花生植株茎秆内分离到1株对花生青枯病菌Ralstonia solanacearum有强拮抗作用的内生细菌,命名为BZ6-1菌株.拮抗试验表明,该菌具有较广抗菌谱,对多种植物病原真菌有较强的抑制作用.形态观察和16S rDNA同源性分析初步鉴定为解淀粉芽孢杆菌Bacillus amyloliquefaciens.该菌的最佳培养基组成为:糖蜜10g·L-1,蛋白胨0.5g·L-1,酵母膏0.5g·L-1.最适发酵条件为pH8.5、装液量50mL/250mL、温度25℃、转速200r·min-1、培养时间21h,在此条件下拮抗圈直径达34.0mm.田间试验结果表明BZ6-1菌株对花生青枯病防效达62.3%.     

蝴蝶兰软腐病中一种新致病菌的分离与鉴定

 Soft rot disease often affects Phalaenopsis amabilis during the growing season. However, the pathogen of the disease is remaining poorly studied. In this study, bacterial strain R1 was isolated from soft rot tissues in Wuhan. The pathogenic, morphological, physiological, biochemical tests and 16S rDNA sequence analysis were carried out. The homology of 16S rDNA sequence between strain R1 and Pseudomonas grimontii was 99.72%, and its physiological and biochemical properties were also similar to those of Pseudomonas grimontiis. All these evidences indicated that strain R1 could be identified as a novel strain of Pseudomonas grimontii. The pathogenicity of the novel isolate was proved according to the Koch's postulates. This is the first report that Pseudomonas grimontii can cause soft rot disease of Phalaenopsis amabilis.     

Molecular Identification of a New Member of the Clover Proliferation Phytoplasma Group (16SrVI) Associated with Centaurea Solstitialis Virescence in Italy

In the United States, yellow starthistle (Centaurea solstitialis) is an annual invasive weed with Mediterranean origins. Malformed plants displaying witches' broom, fasciations, abortion of buds and flower virescence symptoms were observed in central Italy. Attempts to transmit the causal agent from the natural yellow starthistle host to periwinkle by grafting, resulted in typical symptoms of a phytoplasma, i.e. yellowing and shortening of internodes. The detection of phytoplasmas was obtained from both symptomatic yellow starthistle and periwinkle by the specific amplification of their 16S-23S rRNA genes. PCR amplification of extracted DNA from symptomatic plant samples gave a product of expected size. Asymptomatic plants did not give positive results. An amplicon obtained by direct PCR with universal primers P1/P7 was cloned and sequenced. The homology search using CLUSTALW program showed more than 99% similarity with Illinois elm yellows (ILEY) phytoplasma from Illinois (United States) and 97% with Brinjal little leaf (BLL) phytoplasma from India. Digestion of the nested-PCR products with restriction enzymes led to restriction fragment length polymorphism patterns referable to those described for phytoplasmas belonging to the clover proliferation (16S-VI) group. Since this is a previously undescribed disease, the name Centaurea solstitialis virescence has been tentatively assigned to it. This is a new phytoplasma with closest relationships to ILEY and BLL, but distinguishable from them on the basis of 16S rDNA homology, the different associated plant hosts and their geographical origin.