Genes responsible for coronatine synthesis in <Emphasis Type="Italic">Pseudomonas syringae</Emphasis> present in the genome of soft rot bacteria

Primers for the PCR amplification of homologous genes encoding polyketide coronafacic acid and coronafacic ligase in the cells of Pectobacterium atrosepticum SCRI1043 (BX950851) were developed to study the presence of these genes in the genome of Pectobacterium sp. and Dickeya sp. Coronafacic ligase catalyses the formation of coronatine from polyketide coronafacic acid and coronamic acid. Coronatine is a toxin produced by Pseudomonas syringae and is one of the major virulence factors in this bacterium. This study using several strains of P. atrosepticum, P. carotovorum subsp. carotovorum and Dickeya sp. isolated in different countries, indicated that all strains of P. atrosepticum possess genes coding coronafacic acid (cfa gene cluster) and coronafacic ligase (cfl). However, these genes were present only in the genome of five out of 50 tested P. carotovorum subsp. carotovorum strains and two out of 34 strains of Dickeya sp. tested. The PCR products homologous to the sequence of cfa7 and cfl gene fragments were sequenced in order to check the level of homology between genes of P. atrosepticum, P. carotovorum subsp. carotovorum and Dickeya sp. The sequences of the gene fragments amplified from all P. atrosepticum strains were almost identical (100% and 99.97%, respectively). The homology of the sequences obtained for P. atrosepticum and sequences of five P. carotovorum subsp. carotovorum and two Dickeya sp. was lower, between 89.69% to 95.00% for the cfl gene fragment, and about 94% for the cfa7 gene fragment.     

Characterisation of new strains of atrazine-degrading Nocardioides sp. isolated from Japanese riverbed sediment using naturally derived river ecosystem

A Gram-positive bacterial strain able to degrade the herbicide atrazine was isolated using a simple model ecosystem constituted with Japanese riverbed sediment and its associated water (microcosm). Treatment of the water phase of the microcosm with 1 mg litre-1 [ring-14C]atrazine resulted in the rapid degradation of atrazine after a 10 day lag phase period. The [ring-14C]cyanuric acid formed was transiently accumulated as an intermediary metabolite in the water phase and was subsequently mineralised through triazine ring cleavage. Possible atrazine-degrading microbes suspended in the water phase of the microcosm were isolated by the plating method while rapid degradation of atrazine was in progress. Among the 48 strains that were isolated, 47 exhibited atrazine-degrading activity. From these 47 isolates, 12 strains that were randomly selected were found to identically convert atrazine to cyanuric acid via hydroxyatrazine. Polymerase chain reaction (PCR) amplification of the genes corresponding to atrazine degradation revealed that these strains at least carried the genes trzN (atrazine chlorohydrolase from Nocardioides C190) and atzC (N-isopropylammelide isopropyl amidohydrolase from Pseudomonas ADP). Physiological characteristics and 16S rDNA partial sequences of six strains that were further selected strongly suggested that all these isolates originated from the same Nocardioides sp. strain. Additionally, only one isolate could mineralise the triazine ring of cyanuric acid. Based on microscopic observations, this strain appears to be a two-membered microbial consortium consisting of Nocardioides sp. and a Gram-negative bacterium. In conclusion, atrazine biodegradation in the microcosm appeared to occur predominantly by Nocardioides sp. to yield cyanuric acid, which could be mineralised by the other relatively ubiquitous microbes.     

Occurrence and characterization of a Phytophthora sp. pathogenic to asparagus (Asparagus officinalis) in Michigan

A homothallic Phytophthora sp. was recovered from asparagus (Asparagus officinalis) spears, storage roots, crowns, and stems in northwest and central Michigan in 2004 and 2005. Isolates (n = 131) produced ovoid, nonpapillate, noncaducous sporangia 45 microm long x 26 microm wide and amphigynous oospores of 25 to 30 microm diameter. Mycelial growth was optimum at 25 degrees C with no growth at 5 and 30 degrees C. All isolates were sensitive to 100 ppm mefenoxam. Pathogenicity studies confirmed the ability of the isolates to infect asparagus as well as cucurbits. Amplified fragment length polymorphism analysis of 99 isolates revealed identical fingerprints, with 12 clearly resolved fragments present and no clearly resolved polymorphic fragments, suggesting a single clonal lineage. The internal transcribed spacer regions of representative isolates were homologous with a Phytophthora sp. isolated from diseased asparagus in France and a Phytophthora sp. from agave in Australia. Phylogenetic analysis supports the conclusion that the Phytophthora sp. isolated from asparagus in Michigan is a distinct species, and has been named Phytophthora asparagi.     

Pseudomonassp.1-7中甲基对硫磷水解酶基因的克隆及酶学性质研究

为了研究和分离有机磷降解酶及其编码基因,从农药厂污水处理池的活性污泥中分离出一株可以高效降解甲基对硫磷的细菌 1-7, 经16S rDNA鉴定为假单胞菌Pseudomonas sp.。通过构建基因组文库的方法克隆了 1-7 的甲基对硫磷水解酶基因 ophc3, 该基因全长为975 bp,编码324个氨基酸,其中前24个氨基酸残基可能为信号肽序列。将其在大肠杆菌中表达,并对重组甲基对硫磷水解酶(OPHC3)进行纯化和酶学性质的研究结果表明,其酶促反应最适pH值为8.0,在pH 6.0~10.0的范围内放置30 min酶的相对活性均在70%以上;最适反应温度为45 ℃,但该酶不耐高温,60 ℃下保温10 min,相对活性降至46.77%。     

二氯喹啉酸降解菌的分离鉴定及降解特性分析

采用富集培养技术从长期施用二氯喹啉酸的土壤中分离得到1株能够降解二氯喹啉酸的细菌,将其命名为J3,通过生理生化特性和16s rDNA同源性序列分析,鉴定其为产碱菌属(Alcaligenes sp.)。在二氯喹啉酸初始质量浓度为100 mg/L、接种量为4%、pH 7、30 ℃条件下,接种后第6天,菌株J3对二氯喹啉酸的降解率可达到70%以上;在25 ℃、接种量为4%条件下,其生物修复作用可使受二氯喹啉酸(25%田间推荐剂量)药害盆栽烟草的恢复率达到69%。     

Biological control of fusarium seedling blight disease of wheat and barley

ABSTRACT Fusarium fungi, including F. culmorum, cause seedling blight, foot rot, and head blight diseases of cereals, resulting in yield loss. In a screen for potential disease control organisms and agents, Pseudomonas fluorescens strains MKB 100 and MKB 249, P. frederiksbergensis strain 202, Pseudomonas sp. strain MKB 158, and chitosan all significantly reduced the extent of both wheat coleoptile growth retardation and wheat and barley seedling blight caused by F. culmorum (by 53 to 91%). Trichodiene synthase is a Fusarium enzyme necessary for trichothecene mycotoxin biosynthesis; expression of the gene encoding this enzyme in wheat was 33% lower in stem base tissue coinoculated with Pseudomonas sp. strain MKB 158 and F. culmorum than in wheat treated with bacterial culture medium and F. culmorum. When wheat and barley were grown in soil amended with either chitosan, P. fluorescens strain MKB 249, Pseudomonas sp. strain MKB 158, or culture filtrates of these bacteria, the level of disease symptoms on F. culmorum-inoculated stem base tissue (at 12 days post- F. culmorum inoculation) was >/=31% less than the level on F. culmorum-inoculated plants grown in culture medium-amended soil. It seems likely that at least part of the biocontrol activity of these bacteria and chitosan may be due to the induction of systemic disease resistance in host plants. Also, in coinoculation studies, Pseudomonas sp. strain MKB 158 induced the expression of a wheat class III plant peroxidase gene (a pathogenesis-related gene).     

Bt菌株FB的生物学特性及其cry、vip基因型鉴定

菌株FB是1株对小菜蛾Plutella xylostella幼虫具有高毒力的苏云金芽胞杆菌Bacillus thuringiensis （Bt）。本研究通过扫描电子显微镜、大质粒电泳、总蛋白SDS-PAGE及菌株生长特征观察的方式研究了菌株FB特征,克隆得到了基因cry1Ia、cry1Ea、cry1Ab、cry2Ab和vip3Aa全长,依据全基因组测序结果得到了1个cry8基因部分片段,首次在Bt菌株中同时发现基因cry1类和cry8类,这五种基因推导的氨基酸序列与已知基因序列相比,最高相似性分别为99%、98%、99%、100%、99%,而cry8半长基因与已知基因仅为63%。生测结果表明,蛋白Cry1Ia、Cry1Ea和Cry1Ab对小菜蛾幼虫具有较高杀虫活性。     

烟草拮抗链霉菌FT05W基因组测序与几丁质酶家族基因鉴定

链霉菌FT05W是一株对烟草黑胫病等土传真菌病害具有良好拮抗作用的生防放线菌。本研究利用Miseq PE300高通量测序平台对链霉菌FT05W基因组进行序列测定，共获得6914188个reads，通过SPAdes软件对序列进行拼接共得1836个contigs，其基因组全长为7699129 bp，GC比为71%，其中编码基因序列长度为6037181 bp，预测出7434个蛋白编码基因（GenBank登录号：NZ_QGMR00000000），预测编码7323个蛋白。获得的基因注释信息为今后深入开展其在烟草上的生物防治机理研究奠定了重要的基础，有助于推动烟草生防链霉菌功能基因的挖掘与利用，为烟草生防链霉菌FT05W的进一步开发与应用提供理论依据。利用MEGA 6.06构建链霉菌FT05W的几丁质酶家族基因系统发育树，共鉴定出7个几丁质酶家族基因，其中6个为18家族几丁质酶基因，1个为19家族基因。     

安徽桑黄花型萎缩病植原体16S rDNA序列分析及分子检测

 Mulberry yellow dwarf(MYD)disease is an quarantine disease and the causal agent is a phytoplasma.Two pairs of published universal primer, P1/P7 and Rm16F2/Rm16R1, based on the 16S-23S rDNA sequence of phytoplasma and total DNA extracted from infected mulberry tissues were employed for PCR and nested-PCR detection.The results revealed that a phytoplasma-specific 1 830 bp fragment with a G+C content of 46.01% was sequenced(GenBank accession No.GQ249410).The sequence shared 99.7% and 99.8% identity with aster yellows, the representatiive phytoplasma in 16SrI group, and mulberry dwarf phytoplasma classified into subgroup B in 16SrI group and named as the MYD phytoplasma strain Anhui(MYD-Anh).A phylogenetic tree based on 16S rDNA sequences was constructed and showed that MYD-Anh was clustered into 16SrI group.Identity of 16S rDNA sequence between MYD-Anh and mulberry yellow dwarf phytoplasma strain Zhenjiang(MD-zj) was nearly 100%, and they might belong to the same strain.Nested-PCR was used to detect the pathogenic phytoplasma from the differential tissues of mulberry infected with MYD-Anh.The results showed that a phytoplasma-specific 1.4 kb fragment was amplified with total DNA extracted from bark and vein.Nested-PCR was more sensitive than PCR for detecting MYD phytoplasma.     

Lectin-like sequences in genome of Borrelia burgdorferi.

Using degenerative primers designed on the basis of known sequences of lectin genes from different sources a fragment of genomic DNA of Borrelia burgdorferi (strain B31) that contained a lectin-like sequence was isolated, cloned and sequenced. The presence of an open reading frame of 268 amino acids (position 1501-2304 bp) and the computer analysis of the predicted amino acid sequence showed 37% of identity and 75% of homology over region of 25 amino acids with the legume lectin proteins, including erythroagglutinating phytohemagglutinin (PHA-E) and leucoagglutinating phytohemagglutinin (PHA-L). The further analysis of the predicted amino acid sequence showed the presence of another two domains (positions 198-211 and 215-226 aa) consisting of the characteristic conserved sequence motifs for legume lectin proteins. Hemagglutinating activity was detected in lysate of B. burgdorferi (strain B31) spirochete and the affinity to fetuin was determined in a hemagglutination inhibition test. Hemagglutinating activity was also found in a crude lysate of the recombinant clones carrying the fragment of B. burgdorferi genomic DNA. The inhibition of agglutinating activity by fetuin, D-galactosamine and D-mannosamine was determined using the standard procedure of hemagglutination inhibition test with native rabbit red blood cells (RBC).     

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。     

Mutagenesis of beta-1,3-Glucanase Genes in Lysobacter enzymogenes Strain C3 Results in Reduced Biological Control Activity Toward Bipolaris Leaf Spot of Tall Fescue and Pythium Damping-Off of Sugar Beet

ABSTRACT Lysobacter enzymogenes produces extracellular lytic enzymes capable of degrading the cell walls of fungi and oomycetes. Many of these enzymes, including beta-1,3-glucanases, are thought to contribute to the biological control activity expressed by several strains of the species. L. enzymogenes strain C3 produces multiple extracellular beta-1,3-glucanases encoded by the gluA, gluB, and gluC genes. Analysis of the genes indicates they are homologous to previously characterized genes in the related strain N4-7, each sharing >95% amino acid sequence identity to their respective counterparts. The gluA and gluC gene products encode enzymes belonging to family 16 glycosyl hydrolases, whereas gluB encodes an enzyme belonging to family 64. Mutational analysis indicated that the three genes accounted for the total beta-1,3-glucanase activity detected in culture. Strain G123, mutated in all three glucanase genes, was reduced in its ability to grow in a minimal medium containing laminarin as a sole carbon source. Although strain G123 was not affected in antimicrobial activity toward Bipolaris sorokiniana or Pythium ultimum var. ultimum using in vitro assays, it was significantly reduced in biological control activity against Bipolaris leaf spot of tall fescue and Pythium damping-off of sugar beet. These results provide direct supportive evidence for the role of beta-1,3-glucanases in biocontrol activity of L. enzymogenes strain C3.     

甲基对硫磷降解菌PF32的分离鉴定及其降解特性研究

从某农药厂采集污泥,采用驯化富集的方式分离得到1株能以甲基对硫磷为唯一碳源生长的菌株,命名为PF32。根据表型特征 、生理生化特性和16S rRNA基因序列相似性分析,鉴定该菌株为寡养单胞菌Stenotrophomonas sp.。PF32能在24 h内降解浓度为100 mg/L的甲基对硫磷,降解率99%以上。PF32降解甲基对硫磷的最适pH值为7.0,最适温度为30℃,该菌降解甲基对硫磷的速率和起始接种量呈正相关。降解谱试验结果表明,PF32对辛硫磷、倍硫磷、杀螟硫磷、三唑磷、毒死蜱也有较好的降解效果。     

Isolation and characterisation of a bacterial strain degrading the herbicide sulcotrione from an agricultural soil

BACKGROUND: The dissipation kinetics of the herbicide sulcotrione sprayed 4 times on a French soil was studied using a laboratory microcosm approach. An advanced cultivation‐based method was then used to isolate the bacteria responsible for biotransformation of sulcotrione. Chromatographic methods were employed as complementary tools to define its metabolic pathway. RESULTS: Soil microflora was able quickly to biotransform the herbicide (DT₅₀ ≈ 8 days). 2‐Chloro‐4‐mesylbenzoic acid, one of its main metabolites, was clearly detected. However, no accelerated biodegradation process was observed. Eight pure sulcotrione‐resistant strains were isolated, but only one (1OP) was capable of degrading this herbicide with a relatively high efficiency and to use it as a sole source of carbon and energy. In parallel, another sulcotrione‐resistant strain (1TRANS) was shown to be incapable of degrading the herbicide. Amplified ribosomal restriction analysis (ARDRA) and repetitive extragenic palendromic PCR genomic (REP‐PCR) fingerprinting of strains 1OP and 1TRANS gave indistinguishable profiles. CONCLUSION: Sequencing and aligning analysis of 16S rDNA genes of each pure strain revealed identical sequences and a close phylogenetic relationship (99% sequence identity) to Pseudomonas putida. Such physiological and genetic properties of 1OP to metabolise sulcotrione were probably governed by mobile genetic elements in the genome of the bacteria. Copyright © 2011 Society of Chemical Industry     

植物土传病原菌拮抗细菌的筛选与鉴定

从作物根际土壤中分离到1056株细菌，筛选出7个具有较强拮抗活性的菌株。室内测定对水稻纹枯病菌、辣椒疫病菌、瓜果腐霉、油菜菌核病菌、棉花枯萎病菌、茄根腐病菌、棉花黄萎病菌、番茄青枯病菌、甘蓝黑腐病菌等重要土传病原菌有较强拮抗作用；温室盆栽试验对番茄青枯病表现出较好防效，其中以BOH2和OH11效果较为明显，防效分别为90．9％和86．4％。通过形态观察、生理生化试验和16SrDNA序列分析，确定OH11为产酶溶杆菌。BOH3为荧光假单胞菌，其余5个菌株为不同芽孢杆菌。     

小麦白粉菌ADP/ATP载体蛋白基因的克隆及表达特征分析

 小麦白粉病是小麦上的一种重要病害。研究小麦白粉菌致病相关基因及其在致病过程中的作用,对于丰富小麦白粉菌致病的分子机制和筛选防治新靶标具有重要意义。前期转录组测序结果提示一个小麦白粉菌ADP/ATP载体蛋白（ADP/ATP carrier protein,AACP）在小麦与白粉菌互作时上调表达。本研究利用cDNA末端快速克隆技术（rapid\|amplification of cDNA ends,RACE）获得了长945 bp具有完整开放阅读框的小麦白粉菌ADP/ATP carrier基因序列,暂命名为BgtAACPx（GenBank登录号为MF197857）该基因编码314个氨基酸残基,利用相关软件进行生物信息分析,结果表明该蛋白为疏水性,含有6个跨膜区,亚细胞定位在线粒体上。与已有小麦白粉菌AACP蛋白的同源性为97%,是一个新的蛋白,同时构建了与其同源蛋白的遗传进化树。定量结果表明该基因在小麦白粉菌吸器期（48 h）、次生菌丝（72 h）至白粉菌刚产孢子又未形成孢子堆（120 h）这段时期高表达,提示该基因可能参与小麦白粉菌对小麦的致病过程。     

The complete genome of the antifungal bacterium Pseudomonas sp. strain MS82

Journal of Plant Diseases and Protection - The genomic sequence of Pseudomonas sp. strain MS82 isolated from the rhizosphere of a soybean plant is reported and analyzed in relation to its extensive...     

1个小麦NBS类抗病基因同源cDNA序列的克隆与鉴定

 利用cDNA末端快速扩增技术对小麦抗叶锈病近等基因系TcLr35中所获得的抗病基因同源片段S2A2 5'端和3'末端序列进行扩增,并根据拼接序列设计特异性引物,进行全长基因的扩增,获得了1个通读的NBS类抗病同源基因S2A2 cDNA序列,该序列全长为3476 bp,编码866个氨基酸序列。经BLASTp比较,该片段含有NB-ARC保守结构域和多个LRR结构域,与已知植物抗病基因I2C-1、L6、RPS2等相应区域相一致。半定量RT-PCR分析表明,该基因在小麦叶片中为低丰度组成型表达。本研究在TcLr35小麦中成功获得了抗病同源基因,这为最终克隆小麦抗叶锈病基因Lr35奠定了基础。     

内蒙古锡林郭勒天然草原禾本科牧草根际18株固氮细菌的初步分类鉴定

采用16SrDNA测序方法对从内蒙古锡林郭勒天然草原四种主要禾本科牧草根际分离获得的18个固氮菌株进行属水平的鉴定,结果表明：其中7株属于土壤杆菌属（Agrobacterium）、3株属于产碱菌属（Alcali-genes）、1株属于固氮菌属（Azotobacter）、1株属于芽孢杆菌属（Bacillus）、3株属于假...