Impact of maize mucilage on atrazine mineralization and atzC abundance

Soil was amended with maize mucilage, a major rhizodeposit, to study its role on the number of culturable soil micro-organisms, the structure of the bacterial community, atrazine mineralization and atzC abundance. The maximal percentage of atrazine mineralization was lower for mucilage-amended than for water-amended soil. Total culturable soil bacteria and 16S rDNA copy number, measured by RT-PCR, presented similar values and were not significantly (P < 0.05) different among treatments. Mucilage applied at a rate of 70 microg C g(-1) dry soil day(-1) over two weeks did not modify the abundance of the total soil microflora. Global structure of soil bacterial communities revealed by RISA analysis was not modified by maize mucilage amendment. Abundance of atzC sequence was only augmented by mucilage addition at the beginning of the experiment. However, this increase was not sustainable in time, as atzC copy number increased in water-amended soil which, in turn, corresponded with the higher percentage of atrazine mineralization observed in this soil. Maize mucilage amendment alone contributed only to minor changes in the atrazine-degrading community in the studied soil.     

西瓜细菌性果斑病菌的16S rDNA序列分析及特异性引物的设计

 利用细菌16S rDNA基因的通用引物对16个供试菌株进行PCR扩增,把扩增产物进行核苷酸序列测定。将获得的序列与GenBank中相关菌株的16S rDNA序列进行同源性分析。以此设计出检测A.a.c的特异性引物,并利用最大简约法构建了16S rDNA系统演化树。系统演化关系分析表明,6~9号供试菌株的16S rDNA序列与A.a.c标准菌株仅有3个位点的差异,其同源性均在99.8%以上,在构建的系统演化树上,它们聚为同一个族群。利用设计的一对特异性引物(BFB64/65),对各供试菌株进行PCR检测,结果只有A.a.c相关菌株产生扩增条带,产物大小与预期一致。     

秦岭山区无机磷细菌筛选及其Biolog和分子生物学鉴定

为获得具有高解磷活性,可用于微生物肥料研制和生产的无机磷细菌菌株资源,采用平板溶磷圈法,从秦岭山区植物根际土壤样品中筛选获得3株高效无机磷细菌,命名为NO.9、NO.15、NO.17,根据钼锑抗比色法,培养5 d后,检测菌株发酵液,确定3株细菌的可溶性磷含量分别为98.12、80.37 mg/L和104.91 mg/L,微生物量磷含量分别为62.85、53.61、36.59 mg/L。生理生化特征、Biolog和16S rDNA鉴定结果表明,3株细菌均属于革兰氏阴性非肠道菌,NO.9菌株为Inquilinus ginsengisoli,NO.15菌株为醋酸钙不动杆菌(Acinetobacter calcoaceticus),NO.17菌株为荧光假单胞菌(Pseudomonas fluorescens)。     

洋葱伯克霍尔德氏菌株Lyc2的鉴定及对棉苗的防病促生作用

 从烟草根际土壤中分离到一株细菌菌株Lyc2,平皿对峙培养显示该菌可显著抑制多种植物病原真菌菌丝体的生长。温室盆栽试验表明,Lyc2对棉花苗期立枯病的防治效果达到48.8%;水培棉花苗试验表明,Lyc2能显著增加棉苗的鲜重和干重,但对株高的影响不显著。该菌经形态、生理生化试验测定及16S rDNA和ITS序列分析,初步确定为洋葱伯克霍尔德氏菌(Burkholderia cepacia);进一步通过种特异的recA基因序列分析,证明Lyc2菌株属于B. cepacia复合物中基因型IX,B.pyrrocinia。     

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.     

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.     

利用16S rDNA结合gyrA和gyrB基因对生防芽孢杆菌R31的快速鉴定

克隆16S rDNA、DNA促旋酶A亚基编码基因gyrA和B亚基编码基因gyrB对分离自石斛兰（Dendrobiumsp.）叶片的广谱抗真菌生防芽孢杆菌R31进行鉴定。首先通过生理生化测定和克隆16S rDNA序列,分析显示其属于芽孢杆菌属（Bacillus）,但不能准确鉴定到种;然后分别利用克隆的gyrA基因和gyrB基因以及其BLAST分析结果构建系统发育树,最终将该菌株确定为枯草芽孢杆菌（B.subtilis）。结果显示利用16S rDNA与gyrA基因组合可以快速鉴定枯草芽孢杆菌,利用16S rDNA与gyrB基因组合可以快速鉴定枯草芽孢杆菌及其近缘种。     

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.     

金莲花绿变植原体的分子鉴定

本研究对河北省大面积发生的金莲花绿变病的病原进行检测和鉴定。以金莲花叶片的总DNA为模板,使用植原体16S rDNA和核糖体蛋白(ribosomal protein)基因rp的特异性引物进行PCR扩增,在感病金莲花样品中扩增到植原体的16S rDNA(1 432 bp)片段和rp基因(1 240 bp)片段。序列分析发现,获得的16S rDNA和rp基因片段与洋葱黄化植原体Onion yellows phytoplasma(GenBank登录号:AP006628)的相似度最高,分别为99.9%和99.3%,确定金莲花绿变病的病原为植原体,暂命名为金莲花绿变植原体Trollius chinensis virescence phytoplasma。对金莲花绿变植原体的16S rDNA进行虚拟RFLP分析,发现其酶切图谱与16SrⅠ-B亚组的洋葱黄化植原体的参照图谱完全一致,相似系数1.00。16S rDNA和rp基因的系统发育进化树显示,金莲花绿变植原体与16SrⅠ-B亚组的植原体聚为一支,属于植原体16S rⅠ-B亚组。     

桃树根际铁载体产生菌MX-26的分离鉴定及诱变选育

从桃树根际土壤中分离到81株细菌,通过铬天青（chrome azural S,CAS）检测法筛选出一株产铁载体能力较强的细菌菌株MX-26,经形态、生理生化特征鉴定及16S rDNA序列分析,确定MX-26为克雷伯氏菌属产酸克雷伯氏菌Klebsiella oxytoca。以MX-26为出发菌株,对其进行紫外诱变、紫外和氯化锂复合诱变及硫酸二乙酯（DES）诱变,得到菌株的最佳诱变组合：紫外线照射时间为180 s,氯化锂终浓度为1.2%,硫酸二乙酯诱变时间为40 min。诱变获得菌株MX26-18,其产铁载体能力较出发菌株提高了32.9%,且遗传性能较稳定。     

Effects of different parameters on the removal of atrazine in a water environment by Aspergillus oryzae biosorption

Atrazine is the most extensively used herbicide in the agricultural and forestry sectors. Nevertheless, along with the increasing usage amount of Atrazine, its harm exposed gradually, the main problem is its residues in the environment. Microbial adsorption may effectively reduce the pollution caused by atrazine residue in the environment. In this study, a strain of fungi with the function of adsorbing atrazine was selected using microbial screening technology. According to its phenotypic characteristics and 18S rDNA gene sequencing, this strain was of the species genus Aspergillus and was named ECUST-TXZC2018. By studying the dynamic adsorption effect of this strain on atrazine, we found that this strain adsorbed atrazine after 36 hr at pH=5–7, and 20–30°C with more than 70% adsorption. These results demonstrated that ECUST-TXZC2018 had potential application ability to control atrazine residue pollution through the biosorption function.     

海南省木豆丛枝病植原体的分子检测及鉴定

 利用植原体通用引物R16mF2/R16mR1和rp (Ⅱ) F1/rp (Ⅱ) R1对海南木豆丛枝病植原体16S rDNA和部分核糖体蛋白(ribosomal protein,rp)基因序列进行PCR扩增、克隆和测序。获得海南木豆丛枝病植原体16S rDNA基因片段为1430bp,rp基因片段为1170bp。核苷酸同源性比较和系统进化树构建表明,引起海南木豆丛枝病的植原体应属于16SrⅡ组中的亚组ⅲ。本研究首次从分子水平确定了引起我国海南木豆丛枝病的病原物为植原体,明确了其分类地位,为该病害流行学研究和防治提供了理论依据。     

放线菌菌株MY02的鉴定及发酵液中抗真菌活性组分的分离

从东北地区蔬菜保护地土壤中筛选分离到一株放线菌菌株MY02,通过对其形态学特征及16S rDNA序列分析,初步鉴定其为龟裂链霉菌龟裂亚种Streptomyces rimosussub sp.rimosus。该菌株的发酵液具有抗真菌活性。发酵液经离心、过滤、萃取、硅胶柱层析等步骤分离纯化其活性物质,并进行高效液相色谱(HPLC)分析,表明其活性物质中含有两种抗真菌活性组分。对活性组分的正丁醇溶液进行紫外光谱扫描,结果表明该活性组分具有四烯类抗生素的典型特征吸收峰。     

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)。     

樱桃花变绿病植原体的分子鉴定

 植原体(phytoplasma)是一类没有细胞壁,不能人工培养,存在于植物筛管细胞中的类似植物病原细菌的原核生物。迄今为止,世界各地报道的1 000余种植物病害与植原体有关,引起的症状主要包括丛枝、黄化、花变绿、花变叶、花器退化等。     

Degradation of simazine by microorganisms isolated from soils of Spanish olive fields

The capability of the microbial flora isolated from an olive field soil from Andalusia to mineralize simazine has been analyzed. From this soil, a group of bacteria capable of degrading 60 mg simazine litre(-1) in less than a week has been isolated. These microorganisms showed a low capacity for degrading this herbicide to carbon dioxide. When total DNA was isolated from this group of bacteria, we were able to detect by PCR the presence of only the atzC and the trzN genes. Some components of this bacterial population have been identified by sequencing of specific fragments from bacterial 16S rDNA, including Variovorax sp, Pseudoxanthomonas mexicana Thierry et al, Acidovorax sp and Methylopila capsulata Doronina et al. These data suggest that this consortium of bacteria performs an incomplete degradation of the simazine     

Changes in soil microbial community response to precipitation events in a semi-arid steppe of the Xilin River Basin,China

In the context of climate change, precipitation is predicted to become more intense at the global scale. Such change may alter soil microbial communities and the microbially mediated carbon and nitrogen dynamics. In this study, we experimentally repackaged precipitation patterns during the growing season(from June to September) of 2012 in a semi-arid temperate steppe of the Xilin River Basin in Inner Mongolia of China, based on the 60-year growing season precipitation data. Specifically, a total amount of 240 mm simulated precipitation was assigned to experimental plots by taking the following treatments:(1) P6(6 extreme precipitation events, near the 1~(st) percentile);(2) P10(10 extreme precipitation events, near the 5~(th) percentile);(3) P16(16 moderate precipitation events, near the 50~(th) percentile); and(4) P24(24 events, 60-year average precipitation, near the 50~(th) percentile). At the end of the growing season, we analyzed soil microbial community structure and biomass, bacterial abundance, fungal abundance and bacterial composition, by using phospholipid fatty acid(PLFA), real-time quantitative polymerase chain reaction(RT-qPCR) and 16S rRNA gene clone library methods. The extreme precipitation events did not change soil microbial community structure(represented by the ratio of PLFA concentration in fungi to PLFA concentration in bacteria, and the ratio of PLFA concentration in gram-positive bacterial biomass to PLFA concentration in gram-negative bacterial biomass). However, the extreme precipitation events significantly increased soil microbial activity(represented by soil microbial biomass nitrogen and soil bacterial 16S rRNA gene copy numbers). Soil fungal community showed no significant response to precipitation events. According to the redundancy analysis, both soil microbial biomass nitrogen and soil ammonium nitrogen(NH_4-N) were found to be significant in shaping soil microbial community. Acidobacteria, Actinobacteria and Proteobacteria were the dominant phyla in soil bacterial composition, and responded differently to the extreme precipitation events. Based on the results, we concluded that the extreme precipitation events altered the overall soil microbial activity, but did not impact how the processes would occur, since soil microbial community structure remained unchanged.     

广东桉树黄化(丛枝)病植原体分子鉴定与检测

从表现黄化(丛枝)症状的桉树上采集病叶,抽提主脉总DNA,采用植原体通用引物与巢式引物进行PCR和巢式PCR扩增,对扩增产物进行克隆和序列测定,获得了植原体的近全长16S rRNA基因及部分16～23S rRNA基因间隔区序列.序列分析揭示,所获得的序列与已知植原体基因组相应区段的序列高度同源,与柳叶菜变叶植原体(epilobium phyllody)和白腊树丛枝植原体(ash witches'-broom)相应序列(GenBank登录号:AY101386和AY566302)同源率为99.9%,与白腊树黄化植原体(aster yellows BD2)相应序列和番茄巨芽植原体(tomato big bud)相应序列同源率分别为99.6%和99.3%.该序列构建的系统进化树表明,引起我国广州地区桉树黄化(丛枝)病的植原体属于16SrI组(即翠菊黄化组),将其暂命名为桉树黄化(丛枝)植原体广东株系(Eucalyp-tus yellowing and witches'-broom phytoplasma strain Guangdong,EYWB-Gd).建立了桉树植原体巢式PCR检测方法,对疑似病样及桉树组培苗进行了检测,多数疑似病样检测结果为阳性,供试的10株组培苗未发现阳性样品.     

Molecular characterization of a phytoplasma causing Phyllody in Clover and other herbaceous hosts in Northern Italy

Red clover (Trifolium pratense) and Ladino clover (Trifolium repens) plants showing phytoplasma-associated symptoms (yellowing/reddening, virescence and phyllody) have been recovered in Friuli-Venezia Giulia, Italy. Using AluI RFLP analysis of PCR amplified 16S rDNA we showed that the disease can be caused independently by two phylogenetically distinct phytoplasmas. One of them showed the very typical 16S rDNA RFLP pattern of the agent of Clover Phyllody in Canada (CCPh). The 16S rDNA of the other phytoplasma (Italian Clover Phyllody phytoplasma, ICPhp) has been PCR amplified, cloned and sequenced. The sequence revealed high similarity (>98%) with phytoplasmas belonging to the X disease cluster, which includes organisms not reported to cause phyllody on their hosts. The analysis by AluI RFLP of the PCR amplified pathogen 16S rDNA from other herbaceous plants (Crepis biennis, Taraxacum officinale, Leucanthemum vulgare) collected nearby with phytoplasma-associated symptoms showed similar patterns. Southern blot hybridization of their EcoRI digested total DNA revealed identical RFLP patterns, suggesting that the causative agent may be the same organism.Abbreviations PCR Polymerase Chain Reaction - rDNA gene for the small subunit ribosomal RNA - RFLP Restriction Fragment Length Polymorphism     

黑龙江大豆轮作和连作土壤细菌群落多样性比较

为研究大豆轮作和连作对土壤细菌群落多样性的影响,提取黑龙江大豆轮作和连作土壤微生物总DNA,采用细菌通用引物27F和1492R扩增了土壤细菌16S rDNA片段,构建了细菌16S rDNA克隆文库,并对文库中的细菌群落多样性进行了分析。通过16S rDNA序列同源性分析,发现76.5%克隆序列与环境中未培养细菌的16S rDNA序列有较高的相似性,仅有23.5%的克隆序列与GenBank数据库中可培养细菌有较高的相似性,表明黑龙江大豆田土壤中的多数细菌尚未培养。系统发育分析表明黑龙江土壤细菌分属于6大类群,其中变形菌Proteobacteria和酸杆菌Acidobacteria所占比例较大,另有少量厚壁菌Firmicutes、放线菌Actinobacteria、芽单胞菌Gemmatimonadetes和未分类的细菌。大豆轮作土壤细菌多样性更为丰富,并以变形菌为优势细菌类群;而大豆连作土壤细菌多样性有所减少,以酸杆菌为优势细菌类群。表明黑龙江大豆田土壤中细菌多样性十分丰富,其种植方式对土壤细菌群落结构影响较大。