阿特拉津降解菌LY-2的分离鉴定及其对污染土壤的修复

农田土壤中残留的莠去津易对后茬敏感作物造成药害,利用功能微生物调控作物中残留莠去津降解是缓解其药害的有效方式。本研究分析了水稻根际微生物群落及土壤中莠去津降解基因对莠去津胁迫的响应,并从水稻根际土壤中分离出一株莠去津降解菌Pseudomonas sp. AT2,通过盆栽试验探究了莠去津胁迫下菌株AT2对水稻生长及莠去津降解的影响机制。结果表明：莠去津胁迫可显著改变水稻根际细菌的群落结构,并提高根际土壤中莠去津降解基因TrzN、AtzB和AtzC的丰度。莠去津胁迫下,接种降解菌AT2组水稻的株高、根长、干重和叶绿素含量均显著增加,MDA的积累减少,说明AT2可缓解水稻的氧化胁迫损伤;接种降解菌AT2处理组土壤和水稻中莠去津含量分别比对照降低了14.9% 和47.1%~57.5%,说明AT2促进了土壤及水稻中莠去津的降解。另外,莠去津胁迫下,接种AT2还可诱导水稻中莠去津降解基因的上调表达,表达量为未接菌对照组的1.3~2.7倍。研究表明,根际接种降解菌株能激活水稻体内降解基因的表达,促进“土壤-水稻”体系中莠去津的降解,缓解环境中残留莠去津对水稻的毒害作用。

     

Quantification of potato common scab pathogens in soil by quantitative competitive PCR with fluorescent quenching‐based probes

Common scab of potato tubers caused by pathogenic Streptomyces spp. is a cause of serious economic loss worldwide. For the rapid and accurate quantification of pathogenic Streptomyces spp. residing in soil, a new competitive real‐time PCR method using fluorescent quenching‐based probes (quantitative competitive quenching probe PCR: QCQP‐PCR) was developed. The virulence gene of pathogenic Streptomyces spp., nec1, was selected as the target for QCQP‐PCR. A specific primer set to amplify the nec1 gene, and a fluorescently labelled probe that specifically hybridizes with the nec1 amplicon were designed. For QCQP‐PCR, an internal standard DNA (IS DNA) that is identical to the nec1 amplicon but has a 4‐base mismatch in the probe‐hybridizing region, and a fluorescently labelled probe IS, which specifically hybridizes with IS DNA at the mutagenized region, were PCR‐synthesized. The target nec1 gene was co‐amplified with the known copy number of IS DNA by PCR using the same primer set in the presence of the specific probes. The PCR products were monitored in real‐time by measuring the fluorescence intensity (quenching) of each probe. The initial amount of the nec1 gene was quantified based on the ratio of the PCR products of the same PCR cycle. The results revealed that QCQP‐PCR could be used to precisely quantify the nec1 gene, even in the presence of PCR inhibitors in the soil samples examined. The lower limit of quantification was 20 copies per tube, which corresponded to 1500 copies per g dry soil. The quantification achieved by this method was completed within 5 h, i.e. the duration of the entire analysis. These results demonstrate the usefulness of the present method for monitoring pathogenic Streptomyces species in soil.     

莠去津土壤残留生物修复菌肥作用原理与田间应用

莠去津是玉米田应用的优秀除草剂品种,然而由于其在土壤中残留时间长,常对轮作后茬敏感作物造成严重毒害。采用生物修复菌肥做种肥、结合菌肥拌种和叶面喷施方法,研究对玉米后茬旱直播水稻生长发育及药害修复机理,对土壤中莠去津残留量、水稻生长和生理指标、土壤酶活性进行测定。结果表明:颗粒菌肥做种肥+粉剂菌肥拌种+水剂菌肥叶面喷施是莠去津土壤残留毒害修复的最佳方法,土壤中莠去津含量在喷施菌肥后7 d从施用菌肥前的0.9 mg/kg下降到0.1 mg/kg,水稻叶片叶绿素含量显著增加33.74%,超氧化物歧化酶和过氧化物酶酶活性分别显著提高23.39%和92.57%,丙二醛含量则显著降低48.01%;水稻株高、地上部鲜重、干重分别比对照显著增加22.33%、67.51%和74.80%,根系鲜重和干重分别比对照显著增加33.98%和55.43%;土壤磷酸酶、脲酶及纤维素酶含量分别显著增加49.17%、528.65%和35.21%。     

河北省甘薯茎腐病研究初报

甘薯茎腐病是近几年在我国发现的一种新的细菌性病害,目前该病已在我国的福建、广东、江西、广西、海南、河南、重庆、江苏等地发生。2013年10月在对河北省甘薯病害的调查中在文安县发现了大量疑似甘薯茎腐病的病株,给当地的甘薯生产造成了严重的影响。经对疑似病株病原菌的分离纯化、柯赫氏法则验证、形态观察以及基于16SrDNA序列的分析,最终确定该病害为甘薯茎腐病,病原菌为达旦提狄克氏菌(Dickeya dadantii)。这是该病害首次在河北省被发现。     

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。     

Development of a quantitative real‐time PCR assay for Phytophthora infestans and its applicability to leaf,tuber and soil samples

A sensitive real‐time polymerase chain reaction (PCR) assay was developed for the quantification of Phytophthora infestans, the cause of foliar and tuber late blight in potato. A primer pair (PinfTQF/PinfTQR) and a fluorogenic probe (PinfTQPR) were designed to perform a quantitative assay for the detection of P. infestans in leaves, tubers and soils. The assay was shown to be specific to P. infestans and the very closely taxonomically related non‐potato pathogen species P. mirabilis, P. phaseoli and P. ipomoea, but did not detect the potato pathogens P. erythroseptica and P. nicotianae. The assay was able to reliably detect P. infestans DNA at 100 fg per reaction and was effective in quantifying P. infestans in infected leaf tissue from 24 h after inoculation and also in infected symptomless tubers and diseased tubers. Attempts to detect oospores of P. infestans in naturally and artificially infested soil samples are described and compared with baiting tests and previous literature. It was not possible to detect oospores in soil samples due to problems with DNA extraction from the oospores themselves. However, the assay was shown to detect even very low levels of asexual inoculum (sporangia and mycelium) in soil. This work assembles all the necessary features of a quantitative P. infestans assay, which have previously been somewhat disparate: the sensitivity, specificity and quantitation are fully validated, the assay is shown to work in common applications in leaf and tuber tissue and the problems with P. infestans oospore detection are explored and tested experimentally.     

采用16S rDNA鉴定甜瓜细菌性叶斑病菌

从甘肃河西、新疆阿勒泰甜瓜上分离获得的2株致病细菌,通过16S rDNA序列测定以及序列同源性比较,结合病原菌落培养性状、菌体形态观察和革兰氏染色反应等,初步确定当地甜瓜细菌性叶斑病菌为丁香假单胞杆菌[Pseudomonas syringae pv.lachrymans (Smith et Bryan) Younget al.]     

番茄细菌性软腐病病原的分离与鉴定

 近年来,山东省以及浙江省部分地区,番茄陆续出现茎秆严重腐烂现象。发病初期,番茄茎秆、枝条上出现椭圆或条状灰褐色腐斑,然后逐渐扩散、腐烂。湿度大的情况下,植株表皮湿腐,伴随异味产生,茎秆很快腐烂直至整株死亡,给菜农造成严重的经济损失。目前国内对番茄软腐病虽有调查报道,但未见该病原的分离鉴定研究。因此,明确该病原菌,尤其是该病原菌的生理生化及分子生物学特征,为该病害的防治提供可行依据,从而解决生产上的实际问题,显得尤为迫切。     

甘肃番茄细菌性斑点病病原菌鉴定

采自发病番茄植株上分离获得的菌株,经致病性测定、培养特性、革兰氏染色反应、菌体形态、16S rDNA基因序列分析、生理生化以及寄主范围测定,结果表明:甘肃省张掖市番茄细菌性叶斑病菌为丁香假单胞杆菌番茄致病变种[Pseudomonas syringae pv.tomato(Okabe) Young,Dye&;Wikie],为此病的综合防治提供了依据。     

朝鲜蓟细菌性根茎腐烂病病原的分离与鉴定

 在湖南省常德市西洞庭管区种植的朝鲜蓟（Cynara scolumus  L.）上发现了一种新的病害,其症状表现为地上部分从外层叶片开始逐步枯萎,随后根部和主茎杆的髓部腐烂变褐,最后整株枯萎。从田间感病朝鲜蓟茎杆的病健交界处用NA培养基分离,获得10个菌株,分别指定为HNXDT001~010,并进行了致病性测定、形态观察和细菌学特征分析,同时对HNXDT002菌株进行分子生物学鉴定。结果表明,该系列菌株在NA培养基上均形成灰白色圆形菌落,稍突起,有光泽,半透明。在显微镜下菌体呈短杆状,两端钝圆,具有2～8根周生鞭毛,革兰氏阴性。10个菌株通过针刺法接种均可导致朝鲜蓟茎杆、胡萝卜、辣椒、白菜、土豆、番茄和莴苣茎杆软腐,经科赫法则验证为致病病原菌。该菌株的16S rDNA序列和果胶酶基因片段测序(分别用16S rDNA通用引物16SF/16SR和果胶酶基因引物Y1/Y2扩增)与系统发育学分析表明,其16S rDNA序列(GenBank Accession No. JF721958)与胡萝卜软腐果胶杆菌胡萝卜亚种（Pectobacterium carotovorum  subsp.  carotovorum）菌株ATCC15713 (GenBank Accession No. U80197)序列同源性高达99％;果胶酶基因序列(GenBank Accession No. JF721960)与胡萝卜软腐果胶杆菌胡萝卜亚种（Pectobacterium carotovorum subsp. carotovorum）PC1菌株(GenBank Accession No. CP001657)序列同源性为93%。结果表明：朝鲜蓟细菌性根茎腐烂病病原为胡萝卜软腐果胶杆菌胡萝卜亚种。     

Molecular characterization and diagnostics of stubby root and virus vector nematodes of the family Trichodoridae (Nematoda: Triplonchida) using ribosomal RNA genes

Plant parasitic nematodes of the family Trichodoridae cause substantial yield losses in many agricultural crops. Rapid and accurate identification of trichodorids to the species level is critical for selection of appropriate measures for control. This study analysed 99 sequences of the D2–D3 expansion segments of the 28S rRNA gene and 131 sequences of the 18S rRNA gene from the stubby nematodes belonging to the genera Nanidorus, Paratrichodorus and Trichodorus. Species delimiting was based on the integration of morphological identification, which is not provided in the present article, and molecular‐based phylogenetic inference and sequence analysis. Twenty‐two valid species and several species complexes were identified among nematodes included in the analysis. PCR‐RFLPs of the partial 18S rDNA and the D2–D3 expansion segments of the 28S rDNA were tested and proposed for identification of these nematodes. Gel PCR‐RFLP profiles and tables with restriction fragment lengths for several diagnostic enzymes are provided for identification. Some problems of taxonomy and phylogeny of nematodes of the family Trichodoridae are also discussed.     

Detection of Colletotrichum coccodes from soil and potato tubers by conventional and quantitative real-time PCR

Colletotrichum coccodes is the causal agent of the potato blemish disease black dot. Two PCR primer sets were designed to sequences of the ribosomal internal transcribed spacer (ITS1 and ITS2) regions for use in a nested PCR. The genus-specific outer primers (Cc1F1/Cc2R1) were designed to regions common to Colletotrichum spp., and the species-specific nested primers (Cc1NF1/Cc2NR1) were designed to sequences unique to C . coccodes . The primer sets amplified single products of 447 bp (Cc1F1/Cc2R1) and 349 bp (Cc1NF1/Cc2NR1) with DNA extracted from 33 European and North American isolates of C. coccodes. The specificity of primers Cc1NF1/Cc2NR1 was confirmed by the absence of amplified product with DNA of other species representing the six phylogenetic groups of the genus Colletotrichum and 46 other eukaryotic and prokaryotic plant pathogenic species. A rapid procedure for the direct extraction of DNA from soil and potato tubers was used to verify the PCR assay for detecting C. coccodes in environmental samples. The limit of sensitivity of PCR for the specific detection of C. coccodes when inoculum was added to soils was 3·0 spores per g, or the equivalent of 0·06 microsclerotia per g soil, the lowest level of inoculum tested. Colletotrichum coccodes was also detected by PCR in naturally infested soil and from both potato peel and peel extract from infected and apparently healthy tubers. Specific primers and a TaqMan fluorogenic probe were designed to perform quantitative real-time (TaqMan) PCR to obtain the same levels of sensitivity for detection of C. coccodes in soil and tubers during a first-round PCR as with conventional nested PCR and gel electrophoresis. This rapid and quantitative PCR diagnostic assay allows an accurate estimation of tuber and soil contamination by C. coccodes .     

分散固相萃取-超高效液相色谱-串联质谱法同时检测大豆植株及土壤中烟嘧磺隆、莠去津及其代谢物残留

土壤中烟嘧磺隆和莠去津的高残留往往会导致后茬大豆药害问题。本研究建立了一种分散固相萃取-超高效液相色谱-串联质谱法同时检测大豆植株及土壤中烟嘧磺隆、莠去津及其代谢物残留方法。样品经含2%甲酸的乙腈提取, 经分散固相萃取净化, 以乙腈和0.2%甲酸水为流动相, 采用Poroshell 120 EC-C18色谱柱梯度洗脱, 基质匹配标准曲线外标法定量分析。结果表明:在0.01、0.10 mg/kg和1.00 mg/kg添加水平下, 烟嘧磺隆、莠去津及其代谢物的平均回收率为70%~113%, 相对标准偏差为0.3%~11.8%, 目标化合物质量浓度与对应的峰面积之间在0.001~1 mg/kg范围内线性关系良好, 决定系数R2≥0.984 1, 方法的定量限为0.01 mg/kg。该方法简便、快捷、准确, 适用于土壤和幼苗期至鼓粒期大豆植株中烟嘧磺隆、莠去津及其代谢物的检测。本研究为烟嘧磺隆和莠去津的科学使用及后茬作物的安全种植提供有效监测方法。     

Impact of a new biopesticide produced by Paenibacillus sp. strain B2 on the genetic structure and density of soil bacterial communities

The effect of paenimyxin, a new biopesticide produced by Paenibacillus sp. strain B2, on the density of soil bacterial communities was assessed by colony counting and by 16S rDNA and nirK quantitative polymerase chain reaction (PCR). Paenimyxin had a negative effect on the bacterial colony-forming unit (CFU) number, which was significantly reduced 2 and 4 days after treatment. The effect of paenimyxin on cultivatable bacteria was negligible 7 days after treatment. Approximately 10(7) 16S rDNA sequences per gram of soil (dry weight) were detected by quantitative PCR in all samples. Paenimyxin did not affect the quantification of 16S rDNA or of the denitrifying bacterial community. In addition, RISA fingerprinting showed that the genetic structure of the bacterial communities was significantly modified 2 days after paenimyxin application at 50 microM and 4 days after treatment at lower concentrations (0.5 and 5 microM). The impact of paenimyxin treatment on the genetic structure of soil bacterial communities was transient, as no effect could be observed after 7, 14 and 28 days when compared with the untreated control.     

不同施肥模式对盐碱化稻作土壤细菌群落的影响

为了提高盐碱化土壤肥力,确定适宜的施肥模式,以宁夏银北西大滩核心试验站的盐渍化荒地为对象,采用Illumina高通量测序分析法研究了脱硫废弃物与有机肥不同施肥模式1)对照Control(不施脱硫废弃物和有机肥),2) T1处理(脱硫废弃物31 250 kg·hm~(-2)),3) T2处理(31 250 kg·hm~(-2)+有机肥10 000 kg·hm~(-2)),4) T3(31 250kg·hm~(-2)+有机肥18 000 kg·hm~(-2)),5) T4(脱硫废弃物31 250 kg·hm~(-2)+有机肥25 000 kg·hm~(-2))对盐碱化稻作土壤理化性质、细菌群落结构的影响。结果表明,与对照组相比施用脱硫废弃物31 250 kg·hm~(-2)和有机肥25 000 kg·hm~(-2)处理组(T4)显著增加了水稻土壤有机碳(6.74%)、碱解氮(37.20%)、速效磷(47.83%)、铵态氮含量(96.26%)。测序结果表明,土壤中Proteobacteria,Bacteroidetes,Gemmatimonadetes,Actinobacteria,Acidobacteria,Firmicutes,Chloroflexi,Spirochaetes,Euryarchaeota和Nitrospirae为排名前10位的优势菌群,占总分类单元的96.05%～94.32%。主成分分析发现,施用脱硫废弃物和有机肥可以显著的影响细菌群落结构,土壤细菌群落的α多样性指数随着有机肥含量的增加而增加。T4处理组土壤细菌群落结构与其它处理差异显著。在属水平,包括盐单胞菌(Halomonas)、Sulfurimonas以及Lutibacter在内的12个优势细菌属的相对丰度在T4处理组显著增加(P0.05)。RDA分析表明盐碱化土壤碱解氮和铵态氮含量分别解释了水稻土壤细菌群落结构变异的16.9%和14.0%(P0.05)。这些结果均表明短期不同施肥模式下对盐碱化稻作土壤具有一定的改良效果。     

水稻转录因子OsBTF3对不同病原菌和信号分子的基因表达反应

 转录因子基因OsBTF3在水稻品种日本晴悬浮细胞中受白叶枯病菌(Xanthomonas oryzae pv.oryzae,Xoo)诱导表达。为了阐明OsBTF3在水稻叶组织中的表达特征,本研究利用RT-Q-PCR技术,对经3种亲和性病原菌[水稻白叶枯病菌(Xoo)、水稻条斑病菌(Xooc)和稻瘟病菌(Mg)]接种和4种信号分子[脱落酸(ABA)、水杨酸(SA)、茉莉酸甲酯(MeJA)、乙烯(ETH)]诱导处理的水稻叶片中OsBTF3的转录本进行了定量分析。结果表明,OsBTF3对Xoo、Xooc和Mg侵染的基因表达反应均显著地受到诱导,但反应速度和强度略有差异。而4种信号分子对OsBTF3表达的诱导作用差异较大,ABA诱导活性最强,MeJA和ETH次之,SA诱导作用不显著。因此,OsBTF3基因表达不仅具有病原菌Xoo、Xooc和Mg的诱导性,而且也具有信号分子MeJA、ETH和ABA的应答性。     

香蕉枯萎和细菌性软腐病菌的多重PCR检测

香蕉枯萎病菌Fusarium oxysporum f. sp. cubense和细菌性软腐病菌Dickeya zeae的复合侵染为害给香蕉产业发展带来严重挑战, 有必要建立相关病害的多重聚合酶链式反应(multiplex polymerase chain reaction, multiplex PCR)检测技术。本文基于尖孢镰刀菌古巴专化型1号生理小种(F. oxysporum f. sp. cubense race 1, FOC1)基因组contig 438区间(35 631-37 693 bp)(GenBank: AMGP01000438.1)和4号生理小种(F. oxysporum f. sp. cubense race 4, FOC4)基因组contig 195区间(4 028-6 126 bp)(GenBank: AMGQ01000195.1)存在160 bp插入序列差异设计特异扩增引物FOC-F/-R, 同时以香蕉细菌性软腐病菌D. zeae的促旋酶B 亚单位基因(the subunit B of gyrase gene)(GenBank: JQ284039)序列设计特异扩增引物gyrB-F/-R。多重PCR检测结果显示:本技术可在一次PCR扩增反应内同时检测香蕉枯萎病菌1号、4号生理小种和细菌性软腐病菌; 多重PCR的灵敏度结果表明:检测香蕉枯萎病菌的DNA浓度最低限为0.1 ng/μL, 细菌性软腐病菌的灵敏度为10 3cfu/mL;检测结果稳定可靠。因此, 本研究建立的多重PCR检测方法可有效应用于检测香蕉发病组织中的香蕉枯萎病菌和细菌性软腐病菌, 也可用于香蕉种苗和田间土壤带病菌的监测, 为香蕉种植保驾护航。     

Infection of horse chestnut (Aesculus hippocastanum) by Pseudomonas syringae pv. aesculi and its detection by quantitative real-time PCR

Pseudomonas syringae pv. aesculi is a pathogenic bacterium causing bleeding canker disease of horse chestnut ( Aesculus hippocastanum ). This is a serious disease which has been affecting horse chestnut in several European countries over the last five years; however, very little is known about the biology of the causal agent. One of the obstacles to studying this pathogen is the lengthy procedure associated with confirming its presence on the host. In this study, P. syringae pv. aesculi was isolated from lesions on different parts of horse chestnut and its pathogenicity confirmed on horse chestnut saplings using two inoculation techniques. Real-time PCR primers were developed based on gyrase B gene sequence data for the specific detection of P. syringae pv. aesculi . Primer specificity was tested on isolates of the target pathogen as well as on a broad range of related non-target bacteria and other bacterial spp. which inhabit horse chestnut. The real-time primers reliably amplified P. syringae pv. aesculi down to 1 pg of extracted DNA, with and without the presence of host DNA, and also amplified unextracted DNA in whole cells of the bacterium down to at least 160 colony forming units. Detection and quantification of the target pathogen in phloem and xylem of both naturally infected and inoculated horse chestnut tissues was also demonstrated. This quantitative real-time PCR assay provides the facility to study several important aspects of the biology of P. syringae pv. aesculi on horse chestnut including its potential for dissemination in different substrates.     

Real‐time and qualitative PCR for detecting Pseudomonas syringae pv. actinidiae isolates causing recent outbreaks of kiwifruit bacterial canker

Since 2008, Pseudomonas syringae pv. actinidiae virulent strains (Psa‐V) have quickly spread across the main areas of kiwifruit (Actinidia deliciosa and A. chinensis) cultivation causing sudden and re‐emerging outbreaks of bacterial canker to both species. The disease caused by Psa‐V strains is considered worldwide as pandemic. Recently, P. syringae strains (ex Psa‐LV, now called PsD) phylogenetically related to Psa‐V have been isolated from kiwifruit, but cause only minor damage (i.e. leaf spot) to the host. The different biological significance of these bacterial populations affecting kiwifruit highlights the importance of having a diagnostic method able to detect Psa‐V, which is currently solely responsible for the severe damage to the kiwifruit industry. In order to improve the specific molecular detection of Psa‐V, a real‐time PCR assay has been developed based on EvaGreen chemistry, together with a novel qualitative PCR (PCR‐C). Both methods are based on specific primer sets for the hrpW gene of Psa. The real‐time PCR and PCR‐C were highly specific, detecting down to 50 and 200 fg, respectively, and were applied to a range of organs/tissues of kiwifruit with and without symptoms. These methods are important tools for both sanitary and certification programmes, and will help to avoid the spread of Psa‐V and to check possible inoculum sources. In addition to being used as routine tests, they will also enable the study of the biology of Psa‐V and the disease that it causes, whilst avoiding the detection of other populations of related P. syringae present in kiwifruit.