黑龙江省部分地区马铃薯疮痂病菌种类及致病性鉴定

为明确黑龙江省马铃薯疮痂病病原菌的种类及其特征,2012-2013年从黑龙江省克山县、绥化市、哈尔滨市、杜尔伯特蒙古族自治县采集具有疮痂病病斑的马铃薯块茎,从中分离纯化病原菌,根据16SrRNA基因的差异采用分子手段对所分离的菌株进行种类和致病性鉴定,并对txtAB阳性菌株采用萝卜幼苗法或马铃薯致病性试验测定其致病性。共分离出74株菌株,鉴定出致病性菌株26株,其中Streptomyces scabies或S.europaeiscabiei 21株,S.turgidiscabies 3株和S.acidiscabies 2株。所有的致病性菌株中共有4种致病岛基因型,即nec1+/tomA+、nec1-/tomA+、nec1+/tomA-和nec1-/tomA。     

马铃薯疮痂病菌毒素及其致病性的研究

 本文对分离自我国不同地区的马铃薯疮痂病菌产生的毒素进行了研究。通过对不同菌株进行毒素提取、纯化和产毒分析发现各致病菌株均能产生同一类毒素,而非致病菌株均未见毒素产生。毒素的活性检测结果表明,毒素能使马铃薯薯片和萝卜幼苗产生与病菌作用相同的症状,说明毒素是疮痂病菌侵染过程中的主要致病因子。     

瓜类果斑病菌致病基因及靶标基因的预测

哈密瓜果斑病是严重危害瓜类的种传细菌性病害,其病原菌为嗜酸菌属西瓜种(Acidovorax citrulli)。本文首先用Bioedit软件建立瓜类果斑病菌全氨基酸序列本地资源库,运用比较基因组学的方法,通过大量查找嗜酸菌属、欧文氏菌属、假单胞菌属、黄单胞菌属、劳尔氏菌属、土壤杆菌属、木质部小菌属中的致病基因和参考文献中已报道的致病基因,下载相关致病基因的氨基酸序列,与瓜类果斑病菌的全氨基酸序列进行localblast,取E<10-5的为候选基因,得到果斑病菌中与致病性相关的基因,并对未知蛋白进行产物预测。同时,根据已经公布的靶标基因,用同样的比对方法,得到果斑病菌中潜在的靶标基因。本研究通过保守估计,得到77个致病性相关蛋白,预测了其中7个未知蛋白的产物,得到46个靶标蛋白。致病基因和靶标基因的分析预测,对以后研究此类基因提供了指导作用,为更好地防治该病害提供了理论基础。     

马铃薯疮痂病菌在植株和田间的分布与动态分析

为探明马铃薯疮痂病菌在植株和土壤中的分布情况及种群动态变化特点,利用常规PCR和定量PCR(qPCR)技术对不同环境的马铃薯疮痂病株和田间植株不同生育期的土壤样品进行病原菌的定性定量检测.结果 表明,病田、温室盆栽和微型薯苗床中马铃薯疮痂病重度发病植株的根、匍匐茎、块茎、地上茎、叶片等组织样品均可检测到184 bp的疮...     

我国马铃薯疮痂病及其防治研究进展

马铃薯疮痂病(potato common scab)是由放线菌目链霉菌属的链霉菌Streptomyces spp.引起的土传兼种传病害,广泛分布于世界各马铃薯种植区,不仅影响马铃薯的外观品质和销售价格,严重时还会导致马铃薯出苗延迟甚至引起幼苗死亡,造成产量下降,给马铃薯产业带来巨大的经济损失,已经成为全球危害马铃薯生产的第四大病害。2015年我国确立马铃薯主粮化战略,推动了马铃薯产业的发展。近年来,随着种植区域和规模不断扩大,马铃薯疮痂病在我国很多省(自治区)有不同程度的发生,并有逐年扩大和加重的趋势,严重影响商品薯、加工原料薯和种薯的生产,成为制约我国马铃薯生产的主要病害。本文对马铃薯疮痂病症状、发病因素、传播规律、致病机理、分类方法以及我国马铃薯疮痂病发生情况、种类及分布进行归纳,并对马铃薯疮痂病防治措施进行总结,以期为我国马铃薯疮痂病的研究和防治奠定理论基础。     

云南省马铃薯疮痂病致病链霉菌种类组成研究

 为明确云南省马铃薯疮痂病病原菌(Streptomyces spp.)的种类及其生物学特征,自2013年从云南省13个马铃薯主产区采集疮痂病病样,共分离到200株链霉菌,通过温室盆栽致病性试验筛选出67株致病菌。通过形态学、生理生化指标、致病性测定及16S rDNA序列分析对获得的菌株进行鉴定。结果显示,引起云南地区马铃薯疮痂病的病原为10种链霉菌,分别为S. caviscabies、S. anulatus、S. scabies、S. turgidiscabies、S. acidiscabies、S. europaeiscabiei、S. luridiscabiei、S. enissocaesilis、S. griseus和S. aureofaciens。其中S. enissocaesilis和 S. anulatus为优势种群,S. caviscabies、S. anulatus和S. luridiscabiei为国内首次报道的病原菌。因此,认为云南省马铃薯疮痂病菌种类复杂多样。     

义乌市马铃薯疮痂病发生原因及防控措施

马铃薯疮痂病已成为威胁义乌市马铃薯生产的最主要病害。当田间有大量病原菌存在时,该病的发生程度与土壤酸碱度、土壤湿度、品种抗性等因素密切相关。选用抗病品种、水旱轮作、施用酸性肥料、调节土壤酸碱度、利用太阳能杀灭土壤病菌,块茎生长期间保持土壤湿度、选用无病种薯并进行消毒、防治好地下害虫等防控措施,可有效地抑制疮痂病的发生。     

磷酸甘油转移酶基因在水稻白叶枯病菌致病力中的作用研究

稻黄单胞菌稻致病变种(Xanthomonas oryzae pv.oryzae,Xoo)引起的水稻白叶枯病,是水稻上的重要致灾病害之一。前期基因组学分析发现,Xoo菌株PXO99^A的PXO＿04062基因可能编码一个磷酸甘油转移酶,推测与病原菌的致病力有关。本研究采用自杀质粒pK18mobsacB介导的方法,构建了该基因的缺失突变体,并对突变体进行了反式互补。水稻上致病性测定发现,与野生型菌株相比,PXO＿04062基因的缺失突变体DM04062在寄主水稻日本晴上的致病力显著下降,并且其胞外多糖产量、运动性、生物膜形成等均明显降低。进一步采用RNA-seq方法比较突变体转录组的变化,发现突变体中的差异表达基因共有533个,其中包括多个致病相关基因,如胞外多糖合成和细胞运动性等相关基因。与野生型相比,这些基因在突变体中的表达均显著下调。这些结果说明,白叶枯病菌的磷酸甘油转移酶基因是通过影响多种致病力相关因子的合成,来影响病菌在水稻上的致病力。     

柑橘溃疡病菌抗铜相关基因copA和 copB的克隆及原核表达

 以柑橘溃疡病菌DNA为模板,对抗铜相关基因copA和copB进行了PCR扩增和克隆,获得了大小分别为1 782 bp 和1 095 bp的目标片段。构建了这2个基因的原核表达载体 (pET-copA和pET-copB),并在大肠杆菌 [Escherichia coli BL21(DE3)] 中成功诱导表达。利用原核表达的融合蛋白免疫大耳白兔,制备了抗copA和copB原核表达蛋白的多克隆抗体。用间接ELISA法测定了所制备的多克隆抗体的效价均为1∶6 400。用所制备的抗体分别对copA和copB的原核表达产物进行Western blot分析的结果显示,在相应位置产生了较强的免疫反应条带,表明所制备抗体能特异性地与相应的抗原发生免疫反应。     

Genotypic and phenotypic characterization of Streptomyces species associated with potato crops in the central part of Colombia

In Colombia, Streptomyces scabiei (syn. S. scabies) is commonly believed to be the causal organism of scab disease in local potato crops. However, very little is known about this organism and about the diversity and pathogenicity of the Streptomyces species associated with potato crops in Colombia. This study, therefore, aimed to elucidate aspects regarding the diversity of these bacteria associated with potato crops in a particular region of Colombia and evaluate their pathogenicity. We obtained 33 isolates of Streptomyces from netted, superficial and deep-pitted potato scab lesions from two main potato-producing regions in Colombia. Of these, 17 were pathogenic based on in vitro and in planta assays. None of these isolates carried the txtA, txtB, or nec1 genes, commonly associated with pathogenicity in Streptomyces, and characteristic of the pathogenicity island (PAI). We also characterized all isolates based on phenotypic characteristics and analysed their phylogenetic relationships using the 16S rRNA, atpD, recA, rpoB, and trpB genes. The isolates were highly diverse, placed in nine clades with 15 different phenotypes. The 17 pathogenic isolates were placed into three clades, namely S. pratensis, S. xiamenensis, and unknown species. This study is a preliminary investigation towards understanding scab disease in Colombia through the study of both pathogenic and nonpathogenic species present in scab disease lesions in potatoes. Also, this is the first report of Streptomyces species associated with potato tubers in Colombia.     

小麦诱导抗性基因TaWIR1b的克隆及表达分析

全蚀病是小麦上一种重要的土传病害。选育和种植抗病品种是防治小麦全蚀病的根本途径,抗病基因研究是抗病育种的基础性工作。根据基因TaWIR1b(Accession no.M94959.1)的全长序列设计引物扩增‘新农19’的cDNA,获得了完整ORF,编码85个氨基酸残基,比对后发现与TaWIR1b序列同源性达100%。根据获得的TaWIR1b基因全长序列设计定量引物,分析TaWIR1b在全蚀菌胁迫条件下不同互作模式的表达特征。结果表明接种全蚀病菌后抗病小麦品种‘新农19’中TaWIR1b基因被诱导表达,接菌后3d达到峰值143.97,感病品种‘新麦19’中峰值出现在接菌后8d,表达量仅为对照的4.22倍,提示该基因可能参与小麦对全蚀病的抗病过程。     

Phytotoxin Production and Aerial Mycelium Formation by Streptomyces scabies and S. acidiscabies in Vitro

Culture conditions (pH, Ca²⁺, phosphate, and temperature) on phytotoxin production (thaxtomin A and concanamycins A and B) and aerial mycelium formation were examined for two strains each of Streptomyces scabies and S. acidiscabies. Thaxtomin production decreased at pH 7.5 and increased at 15°C in S. scabies and increased at 30 mM phosphate in S. acidiscabies. Concanamycin production was stimulated by the addition of CaCl₂ and was correlated with formation of aerial mycelium. Aerial mycelium formation in S. scabies was greater the higher the pH, Ca²⁺ concentration and temperature, whereas that in S. acidiscabies was unaffected. Received 2 November 2000/ Accepted in revised form 27 April 2001     

Novel in vivo use of a polyvalent Streptomyces phage to disinfest Streptomyces scabies-infected seed potatoes

A highly virulent and polyvalent Streptomyces phage was isolated from a potato field near Albany, Western Australia. The efficacy of the isolated phage to disinfest seed potato tubers artificially inoculated with a common scab-causing streptomycete was evaluated. The phage suspension was prepared in a mini-bioreactor. Diseased potatoes were bathed in a phage suspension (1 × 10⁹ plaque-forming units per mL) for 24 h. The suspension was constantly circulated within a novel 25 L phage bath by means of an air-sparging pipe driven from an air compressor. Phage-treated scab-affected seed potatoes planted into free-draining polystyrene boxes containing steam-pasteurized field soil produced tuber progeny with significantly ( P  < 0·05) reduced levels of surface lesions of scab (1·2%) compared with tubers harvested from nonphage-treated tubers (23%). The number of scab lesions was also significantly reduced ( P  < 0·05) by phage treatment of mother tubers. No significant differences were recorded in weight, size or number of harvested tubers from phage-treated or nontreated mother tubers. This is the first in vivo study that has used Streptomyces phage to significantly disinfest seed potatoes of Streptomyces scabies and thereby reduce contamination of soil from seed-tuber-borne inoculum and reduce infection of daughter tubers.     

黄蓝状菌几丁质酶基因tfchi1的克隆、表达及抗菌活性

 为研究黄蓝状菌（Talaromyces flavus）几丁质酶性质和作用,通过RT PCR、3′ RACE及5′ TAIL PCR技术克隆了一个黄蓝状菌几丁质酶基因tfchi1,该基因全长为2 561 bp,含有6个内含子,包含一个1 194 bp的ORF,编码397个氨基酸,分子量为43.47 kDa,属于糖苷水解酶第18家族。利用基因重组的方法构建酵母分泌型表达载体pPIC9K/tfchi1,并转化毕赤酵母,筛选得到一株高效表达几丁质酶的工程菌GS TFCHI1 9,甲醇诱导第7 d酶活性最高,达32.29 U·mL-1。SDS PAGE检测纯化重组蛋白的分子量约44 kDa。重组几丁质酶Tfchi1最适反应温度为35℃,最适反应pH值为5.5,在pH 6～8之间稳定。Zn2+、Cu2+对 Tfchi1活性有明显的抑制作用。抑菌活性测定结果显示,Tfchi1可明显抑制敏感植物病原真菌的菌丝生长和分生孢子萌发。该酶在几丁质资源的开发利用和生物防治等领域具有较广的应用前景。     

荻草谷网蚜ABCG1基因的克隆及表达模式分析

荻草谷网蚜Sitobion miscanthi是严重威胁我国小麦生产安全的迁飞性害虫。蜕皮激素是参与蚜虫翅型分化调控的内激素, 在有翅成蚜体内保持高滴度, 且诱导后代产生更高比例的无翅蚜, 其进出靶细胞需要经过细胞膜上特定蛋白的转运。ATP结合盒转运蛋白家族G亚家族(ATP-binding cassette transporter G, ABCG)中的 ABCG1是通过跨膜转运昆虫类固醇、对蜕皮激素信号进行负调控的功能蛋白之一, 在蚜虫中尚未见报道。本文克隆了荻草谷网蚜ABCG1(SmisABCG1)基因, 并进行了序列比对、系统进化分析以及不同组织部位和发育时期表达模式分析。结果显示, SmisABCG1基因的开放阅读框全长为1 851 bp, 编码616个氨基酸, 含7个跨膜结构域, 符合ABCG蛋白家族典型结构特性, 基因登录ID:OP626323。昆虫间ABCG1较保守, 该蛋白系统进化关系与各自物种间亲缘关系的远近保持一致。其中, SmisABCG1与来自豌豆蚜、禾谷缢管蚜、棉蚜、花生蚜和雪松长足大蚜等的ABCG1氨基酸序列高度一致(>87%), 以上蚜虫聚为一支。与SmisABCG1亲缘关系最近的是豌豆蚜的ABCG1, 其次是半翅目的褐飞虱、白背飞虱和灰飞虱, 与膜翅目的新疆菜叶蜂、阿里山潜蝇茧蜂以及鞘翅目的赤拟谷盗、蜂箱小甲虫亲缘关系较远。该基因在伪胚胎和成蚜阶段高表达。包含伪胚胎的有翅、无翅成蚜整蚜SmisABCG1的转录水平无显著差异, 但其在来自有翅成蚜的伪胚胎中的转录水平高于无翅成蚜伪胚胎, 证实无翅成蚜自身的转录水平较高, 而有翅成蚜较低。进一步分析显示这一差异主要是无翅蚜胸部显著高表达所导致。基于该蛋白对蜕皮激素负调控, 与有翅成蚜转录水平低, 但蜕皮激素水平更高相符合。     

菌寄生真菌纤细齿梗孢一丝氨酸-苏氨酸蛋白激酶基因OPK1的克隆及序列分析

 Using degenerate PCR and TAIL-PCR, a protein kinase gene OPK1 (Genebank accession No.:EU417815) was cloned from mycoparasite fungi Olpitrichum tenellum. OPK1 has an open reading frame of 2 031 bp interrupted by two introns (108 bp, 84 bp) and putatively encodes a protein of 676 aa. Phylogenetic analysis indicated that OPK1 was most similar to other serine-threonine protein kinase in fungi. Southern blot analysis indicated that OPK1 is present as a single copy in the genome. RT-PCR showed it could be transcribed both in the phase of spore germination and hyphal growth.     

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.     

水稻热休克蛋白HSP70基因克隆、表达分析及原核表达

为明确水稻热休克蛋白70（heat shock protein 70,HSP70）基因在逆境胁迫下的表达特征,以日本晴粳稻水稻品种为材料,通过基因克隆方法获得水稻HSP70基因cDNA全长序列,并采用生物信息学的方法分析其序列特征,利用实时荧光定量PCR（quantitative real-time PCR,RT-qPCR）技术测定不同水稻组织、不同激素、不同温度以及不同逆境下HSP70基因的相对表达量,同时对其原核表达条件进行优化。结果表明,水稻HSP70基因全长1 950 bp,预测蛋白大小为71.18 kD,等电点为5.10,亚细胞定位于细胞质内,有较强的亲水性。水稻HSP70蛋白序列与香蕉HSP70蛋白序列的亲缘性较高;水稻HSP70基因表达具有组织特异性,在茎中的相对表达量最高,在根中最低;低温胁迫后,水稻HSP70基因在12 h内基因相对表达量相对稳定,在24 h时相对表达量达到峰值,为9.60;高温胁迫后,在较短时间内水稻HSP70基因显著上调并达到峰值,为79.10;激素吲哚乙酸、脱落酸和油菜素内酯均能诱导水稻HSP70基因表达;但在甘露醇模拟干旱和NaCl盐胁迫处理下,水稻HSP70基因的相对表达量呈现先升后降的趋势。水稻HSP70蛋白的最佳诱导温度是30℃,异丙基-β-D-硫代吡喃半乳糖苷最佳诱导浓度为1.2 mmol/L。     

西藏一种芥菜型油菜PGIP基因的克隆与序列分析

防治油菜菌核病的有效方法是培育抗菌核病的油菜品种。多聚半乳糖醛酸酶阻遏蛋白(polygalacturonase inhibiting protein,PGIP),是植物体内一种重要的抗真菌蛋白。本研究根据NCBI中发表的PGIP基因序列设计引物,从芥菜型油菜‘藏油6号’中克隆得到全长1 048bp的PGIP基因序列。其核酸序列与NCBI数据库中登录的PGIP基因序列同源性为99%。该序列翻译后得到的氨基酸序列与NCBI上公布的油菜PGIP序列同源性为99%。该蛋白质序列中包含8个亮氨酸重复区(leucine-rich repeat,LRR)。过去研究结果显示芥菜型油菜比甘蓝型、白菜型油菜对菌核病的抗性普遍较高,但是未能搞清楚具体的原因,只是提出抗菌核病基因主要分布在芥菜型油菜中,其次是甘蓝型油菜中。通过研究证实了芥菜型油菜上PGIP基因具有保守性、疏水性,而且结构稳定,具有多个信号肽,能够高效地发挥其抗核盘菌的功能。今后在油菜育种上应利用芥菜型油菜的优势,发挥其抗病育种的潜力。