谷子抵御白发病菌侵染的生理生化及基因表达分析

白发病已成为我国谷子生产上的重要病害,不仅造成产量下降,还对谷子品质构成威胁。有关谷子品种响应白发病菌侵染的生理生化机制研究尚未见报道。本研究采用病菌卵孢子和谷粒混合的接菌方法分别接种抗病品种‘G1’和高感品种‘晋谷21’(JG21),并测定抗感谷子的植株形态指标、光合参数、防御酶活性、抗氧化酶活性、可溶性糖及脯氨酸含量,同时结合基因表达分析,旨在明确谷子响应白发病菌侵染机制。结果表明,白发病菌侵染抗感品种后,植株高度及叶面积均表现出不同程度的下降,感病品种的株高降低幅度远大于抗病品种;与抗病品种相比,感病品种中除了胞间CO₂浓度显著升高外,其他光合参数显著下降,且均低于抗病品种。抗病品种中抗氧化酶和防御酶活性及脯氨酸、可溶性糖含量均升高,显著高于感病品种。抗病相关基因Seita.2G024600(PR1)、Seita.7G168700(PAL)、Seita.1G240200(PAL)和Seita.9G462500(POD)在抗病品种中上调,而在感病品种中表达受到抑制或下调。以上光合参数、酶活性、Pro和可溶性糖含量变化等生理生化指标可作为抗性鉴定和种质筛选的参考...     

谷子抵御白发病菌侵染的生理生化及基因表达分析

 白发病已成为我国谷子生产上的重要病害,不仅造成产量下降,还对谷子品质构成威胁。有关谷子品种响应白发病菌侵染的生理生化机制研究尚未见报道。本研究采用病菌卵孢子和谷粒混合的接菌方法分别接种抗病品种‘G1'和高感品种‘晋谷21'(JG21),并测定抗感谷子的植株形态指标、光合参数、防御酶活性、抗氧化酶活性、可溶性糖及脯氨酸含量,同时结合基因表达分析,旨在明确谷子响应白发病菌侵染机制。结果表明,白发病菌侵染抗感品种后,植株高度及叶面积均表现出不同程度的下降,感病品种的株高降低幅度远大于抗病品种;与抗病品种相比,感病品种中除了胞间CO₂浓度显著升高外,其他光合参数显著下降,且均低于抗病品种。抗病品种中抗氧化酶和防御酶活性及脯氨酸、可溶性糖含量均升高,显著高于感病品种。抗病相关基因Seita.2G024600(PR1)、Seita.7G168700(PAL)、Seita.1G240200(PAL)和 Seita.9G462500(POD)在抗病品种中上调,而在感病品种中表达受到抑制或下调。以上光合参数、酶活性、Pro和可溶性糖含量变化等生理生化指标可作为抗性鉴定和种质筛选的参考,这些差异表达基因可能参与了谷子对白发病菌抗病调控。研究结果可为谷子抗病分子机制及分子改良育种提供理论基础。     

辽宁五味子种子带菌检测及药剂消毒处理研究

采用PDA平板检测法对辽宁省6个地区的五味子种子进行带菌检测,并测定了6种杀菌剂对五味子种子的消毒效果。结果表明,五味子种子表面携带的主要真菌类群为曲霉属和青霉属,种子内部寄藏真菌主要包括曲霉属、青霉属、镰刀菌属、根霉属和链格孢属等。供试的6种杀菌剂对五味子种子均有一定的消毒效果,福美双对五味子种子消毒处理效果最显著。     

红花种子带菌检测及药剂消毒处理

采用平皿法检测了红花种子的带菌情况,并研究了5种杀菌剂对红花种子的消毒效果。结果表明,红花种子携带的主要真菌类群为链格孢属(Alternaria spp.)、黄曲霉属(Aspergillus spp.)、镰孢霉属(Fusarium spp.)、黑根霉属(Rhizopus spp.)和青霉菌属(Penicillium spp.)。福美双与多菌灵混剂、噁霉灵、甲基立枯磷、种衣剂1号、种衣剂2号分别对不同批次红花种子所带真菌均具有一定的消毒效果。     

西瓜种子带菌检测及杀菌剂消毒处理效果

采用离体平皿法对来自新疆等4个省区的14个西瓜品种进行种子带菌检测、分离纯化和鉴定,并测定了7种杀菌剂和1种种衣剂对种子带菌的消毒处理效果。结果表明,种子表面携带的优势菌群主要为青霉属Penicillium spp.、根霉属Rhizopus spp.、曲霉属Aspergillus spp.、交链孢属Alternaria spp.和镰孢属Fusarium spp.;种子内部寄藏真菌主要为青霉属、根霉属和曲霉属;不同品种之间种子表面携带真菌种类差异较大,种子内部寄藏真菌种类差异不明显;种壳带菌率一般高于种仁带菌率。15% FDDF ·霜·福悬浮种衣剂、福美双和代森锰锌对种子带菌消毒效果优于多菌灵、敌磺钠、 FDDF 霉灵、拌种灵和甲霜灵。     

甘草种子带菌检测及药剂消毒处理效果

采用PDA平板检测法分别对来自内蒙古、新疆、宁夏和甘肃等10个地区的甘草种子进行带菌检测。结果表明,甘草种子表面携带的优势菌群主要是曲霉属、青霉属和根霉属,其中来自于内蒙古鄂托克前旗和新疆阿勒泰甘草种子镰刀菌的分离频率达到11.1%和1.9%;种子内部寄藏真菌主要是曲霉属、青霉属、根霉属和交链孢属。来自于内蒙古鄂尔多斯杭锦旗和翁都特旗两个地区的甘草种子镰刀菌的带菌率分别达到5.6%和14.2%,其他来源的种子都未检测到镰刀菌属真菌。同时检测了来自于10个不同地区硬实种子的带菌情况,结果显示只有3个地区的硬实种子带菌,带菌率是0～2.5%。福美双、口恶霉灵、甲霜灵、苯醚甲环唑、百菌清和嘧菌酯几种药剂对甘草种子带菌消毒处理具有良好效果,种衣剂咯菌腈抑菌效果显著。     

大豆疫病的检疫研究——种子带菌及检验技术

本文通过人工接菌和对自然条件下的大豆疫病种子的研究，证实大豆疫病可以种子带菌，并以卵孢子和菌丝体存在于种子的种皮、胚和子叶里，且卵孢子只产生于种皮。提出大豆疫病种子检验只需检查种皮，以种皮里大豆疫病卵孢子存在与否为标准。采用ＭＴＴ染色法测定种子带菌卵孢子活性，检查出存放３年的病种子里活性卵孢子占３０％。     

青稞种子带菌检测及杀菌剂消毒处理效果

为了研究青稞种子外部和内部携带真菌情况,比较不同杀菌剂对青稞种子的带菌消毒效果和对幼苗生长的影响,为青稞种子播前包衣处理和种传真菌病害防控提供依据,采用离体平皿法对云南迪庆‘云青1号’、‘云青2号’和‘短白青稞’3个主栽品种进行带菌检测,并对种子进行拌种或浸种处理测定6种杀菌剂对种子消毒效果,分析杀菌剂对种子发芽和幼苗生长的影响。结果表明:供试青稞种子表面携带的优势菌群为青霉(Penicilliumspp.)、镰刀菌(Fusariumspp.);种子内部寄藏的真菌主要为镰刀菌、核腔菌(Pyrenophoraspp.)、附球菌(Epicoccumspp.)、丝核菌(Rhizoctoniaspp.)、链格孢(Alternariaspp.)和木霉(Trichoderma spp.)。青稞不同品种的种子表面及内部携带的真菌种类差异较大。致病性测定表明,镰刀菌对种子萌发和幼苗生长影响最大,后期出现幼苗坏死现象。45%咪鲜胺EW、75%百菌清WP、50%福美双WP对青稞种子携带真菌均有显著抑制作用和消毒效果,50%福美双WP消毒效果最优,达100%;45%咪鲜胺EW、75%百菌清WP、50%福美双WP处理对青稞种子发芽和幼苗生长均无显著影响。     

谷瘟病菌无毒基因AVR1-CO39序列变异及遗传多样性分析

 谷瘟病是谷子上毁灭性病害之一,为了探讨不同地区谷瘟病菌群体的遗传多样性,对我国11个省(自治区)171株谷瘟病菌的无毒基因AVR1-CO39进行扩增测序,并利用ClustalX2.0和DnaSP5.0软件对测序结果进行分析。结果表明,171株谷瘟病菌单孢菌株AVR1-CO39的CDS编码区共有40个多态性位点,依据序列之间的核苷酸差异划分为37个单倍型,H1型为绝对优势单倍型。我国11个省份谷瘟病菌群体的AVR1-CO39具有较高的遗传多态性,由于存在较为频繁的基因交流,种群之间没有明显的遗传分化。种群内部的遗传分化是谷瘟病菌遗传分化的主要方式,错配分布检测结果显示进化过程中可能出现群体扩张,并且谷瘟病菌的聚类与地理来源没有显著的关系。研究结果表明,谷瘟病菌无毒基因AVR1-CO39具有较高的变异性,以H1为核心单倍型在不断地变异衍生出新的等位基因类型,并且这种变异衍生趋势并不受地理隔离的影响。研究结果可为谷子抗病品种选育,揭示谷瘟病菌无毒基因与谷子抗病基因之间的互作机制提供理论支持。     

北京地区主栽西瓜品种种子携带真菌检测及鉴定

用离体平皿法对北京西瓜产区主栽的11个西瓜品种进行种子带菌检测、分离纯化和形态学、ITS序列比对,确定其属或种地位。结果表明:不同品种所带真菌种类有差异,种子表面携带的优势菌群主要为曲霉属Aspergillus spp.、青霉属Penicilliumspp.、链格孢属Alternariaspp.和镰刀菌属Fusariumspp.;种子内部寄藏真菌主要为青霉属、根霉属Rhizopus spp.和曲霉属。种子外部检出的主要病原菌有镰刀菌属的F.verticillioides,F.proliferatum及茎点霉Phomasp.;种子内部检出的病原菌有F.verticillioides和F.oxysporum。不同品种间种子表面携带真菌量与种子内部携带真菌率差异显著,但种子外部带菌量和内部种仁带菌率之间无显著相关性。本研究对开展西瓜种子处理研究有借鉴意义。     

草间钻头蛛共生菌多样性测定及沃尔巴克氏体感染对宿主发育历期和性比的影响

为了解草间钻头蛛Hylyphantes graminicola体内共生菌的组成及共生菌对宿主的影响,通过高通量测序技术确定其体内的优势共生菌,采用常规PCR法对草间钻头蛛进行优势共生菌的感染检测,并研究了优势共生菌感染对草间钻头蛛子代的发育历期和性比的影响。结果表明:经高通量测序细菌16S r DNA的V3、V4区,确定草间钻头蛛的优势共生菌为沃尔巴克氏体Wolbachia和Cardinium。167头被检测蜘蛛中,Cardinium的感染率为100.00%,沃尔巴克氏体的感染率为38.92%。感染沃尔巴克氏体的草间钻头蛛F1代雌蛛的2龄期、3龄期、4龄期和总历期均极显著低于未感染的草间钻头蛛,感染沃尔巴克氏体的草间钻头蛛F1代雌蛛1龄期显著低于未感染的F1代个体,感染和未感染沃尔巴克氏体的草间钻头蛛F1代雌蛛的卵期和5龄期均无显著差异。感染沃尔巴克氏体后,草间钻头蛛F1代雄蛛的1龄期和总历期极显著缩短,2龄期、3龄期和5龄期显著缩短,而卵期和4龄期与未感染沃尔巴克氏体的F1代雄蛛之间无显著差异。另外,沃尔巴克氏体感染极显著提高了草间钻头蛛的子代性比。     

Resistance of wheat to Mycosphaerella graminicola involves early and late peaks of gene expression

Large-scale cDNA-AFLP profiling identified numerous genes with increased expression during the resistance response of wheat to the Septoria tritici blotch fungus, Mycosphaerella graminicola. To test whether these genes were associated with resistance responses, primers were designed for the 14 that were most strongly up-regulated, and their levels of expression were measured at 12 time points from 0 to 27 days after inoculation (DAI) in two resistant and two susceptible cultivars of wheat by real-time quantitative polymerase chain reaction. None of these genes was expressed constitutively in the resistant wheat cultivars. Instead, infection of wheat by M. graminicola induced changes in expression of each gene in both resistant and susceptible cultivars over time. The four genes chitinase, phenylalanine ammonia lyase, pathogenesis-related protein PR-1, and peroxidase were induced from about 10- to 60-fold at early stages (3 h–1 DAI) during the incompatible interactions but were not expressed at later time points. Nine other genes (ATPase, brassinosteroid-6-oxidase, peptidylprolyl isomerase, peroxidase 2, 40S ribosomal protein, ADP-glucose pyrophosphorylase, putative protease inhibitor, methionine sulfoxide reductase, and an RNase S-like protein precursor) had bimodal patterns with both early (1–3 DAI) and late (12–24 DAI) peaks of expression in at least one of the resistant cultivars, but low if any induction in the two susceptible cultivars. The remaining gene (a serine carboxypeptidase) had a trimodal pattern of expression in the resistant cultivar Tadinia. These results indicate that the resistance response of wheat to M. graminicola is not completed during the first 24 h after contact with the pathogen, as thought previously, but instead can extend into the period from 18 to 24 DAI when fungal growth increases dramatically in compatible interactions. Many of these genes have a possible function in signal transduction or possibly as regulatory elements. Expression of the PR-1 gene at 12 h after inoculation was much higher in resistant compared to susceptible recombinant-inbred lines (RILs) segregating for the Stb4 and Stb8 genes for resistance. Therefore, analysis of gene expression could provide a faster method for separating resistant from susceptible lines in research programs. Significant differential expression patterns of the defense-related genes between the resistant and susceptible wheat cultivars and RILs after inoculation with M. graminicola suggest that these genes may play a major role in the resistance mechanisms of wheat.     

Identification and in planta detection of Pseudomonas syringae pv. tomato using PCR amplification of hrpZPst

A rapid detection method based on PCR amplification of Pseudomonas syringae pv. tomato chromosomal sequences was developed. Primer design was based on the P. syringae DC3000 hrpZ_Pst gene, which maps on a pathogenicity-associated operon of the hrp/hrc pathogenicity island.A 532 bp product corresponding to an internal fragment of hrpZ_Pst was amplified from 50 isolates of P. syringae pv. tomato belonging to a geographically representative collection. The amplification product was also obtained from three coronatine-deficient strains of P. syringae pv. tomato.On the other hand, PCR did not produce any such products from 100 pathogenic and symbiotic bacterial strains of the genera Pseudomonas, Xanthomonas, Erwinia, and Rhizobium and 75 unidentified bacterial saprophytes isolated from tomato plants. The method was tested using leaf and fruit spots from naturally-infected tomato plants and asymptomatic nursery plants and artificially contaminated tomato seeds. The results confirmed the high specificity observed using pure cultures.     

Unsaturated fatty acids from zoospores of <Emphasis Type="Italic">Sclerospora graminicola</Emphasis> induce resistance in pearl millet

Downy mildew of pearl millet, caused by Sclerospora graminicola, is a devastating disease, resulting in high economic losses in the semi-arid regions of the world. Recently, induction of host plant resistance using biotic and abiotic inducers are included among disease management practices as an eco-friendly approach. Unsaturated fatty acids are considered as a new generation of plant disease resistance inducers. In the present study, six unsaturated fatty acids, viz. docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA), arachidonic acid (AA), linolenic acid, linoleic acid and oleic acid, all originally detected in the zoospores of S. graminicola,were applied to seeds of susceptible cultivars of pearl millet to examine their ability to protect against downy mildew under greenhouse and field conditions. In greenhouse experiments, EPA and AA induced a maximum of 78.6% and 76.5% protection, whereas linoleic acid, DHA and linolenic acid provided up to 66.3%, 61.2% and 24.5% protection, respectively. Oleic acid was not effective in protecting pearl millet (only 5.1% protection). A time interval of four days between treatment of seeds and challenge inoculation was required to obtain optimum protection. Plants raised from treated seeds and challenge inoculated at the tillering and inflorescence stages showed enhanced resistance, resulting in higher grain yield compared to untreated plants of the same cultivar. Chitinase activity was found to be higher in susceptible seedlings of pearl millet after treatment with the fatty acids and pathogen inoculation than in seedlings only inoculated with the pathogen. This indicates that host defence responses are activated and thus that induced resistance is involved in the protection observed. The role of unsaturated fatty acids as activators of resistance against downy mildew in pearl millet is discussed.     

糜子丝黑穗病病原菌鉴定及其生物学特性

为明确陕西省糜子丝黑穗病病原菌种类及其生物学特性,采用组织分离法和单孢分离法对其进行分离纯化,通过形态学特征和ITS序列分析对其进行鉴定,采用幼苗浸种法测定其致病性,并于室内对其生物学特性进行研究。结果表明,从发病糜子叶片和病瘿中共分离得到6株形态特征一致的菌株,代表菌株YL11-1菌落呈酵母状,白色,厚垣饱子呈球形或近球形,大小约7～10μm;菌株YL11-1的ITS序列(GenBank登录号为KT721292.1)与稷光孢堆黑粉菌Sporisorium destruens的相似性为100%,接种菌株YL11-1后糜子出现4种典型病症,根据形态学特征、生物学特性和致病性分析结果将该病原菌鉴定为稷光孢堆黑粉菌。该菌株厚垣孢子萌发及菌丝最适生长温度范围均为25～30℃,最适产孢温度为30℃;厚垣孢子萌发和菌丝生长最适pH范围分别为3～7和5～7,最适产孢pH为7;厚垣孢子萌发最适碳源为0.5%葡萄糖和1.0%蔗糖,该菌株对葡萄糖的利用效果最好,对淀粉的利用效果最差;菌丝生长和产孢最适氮源分别为硝酸钾和硫酸铵,该菌株对硝酸铵的利用效果最差。     

草间钻头蛛体内沃尔巴克氏体的去除及其感染对宿主生殖和适合度的影响

采用在草间钻头蛛Hylyphantes graminicola的饮水和人工饲料中添加四环素的方法研究了蜘蛛体内沃尔巴克氏体(Wolbachia)的去除.同时,通过设置草间钻头蛛Wolbachia感染种群与未感染种群之间不同的交配方式,研究Wolbachia感染对草间钻头蛛生殖和适合度的影响.结果显示,在草间钻头蛛的饮水和人工饲料中添加四环素均能部分去除草间钻头蛛体内的Wolbachia,在供试最高浓度下,去除率分别为40％和50％.4种不同的交配组合下,草间钻头蛛雌蛛均能产卵,卵的孵化率差异不显著,Wolbachia感染对草间钻头蛛的生殖无影响.感染Wolbachia的草间钻头蛛所产第1卵袋的卵历期为6.65d,极显著高于未感染Wolbachia的卵历期(P =0.008).感染与未感染Wolbachia的草间钻头蛛种群世代存活率、性比以及适合度均无显著差异.     

氯虫苯甲酰胺和氟虫腈对黏虫细胞色素P450基因表达的影响

为筛选出黏虫Mythimna separata参与杀虫剂解毒代谢的主效细胞色素P450基因,采用叶片浸渍法测定了用于处理黏虫3龄幼虫的亚致死浓度,通过构建转录组测序文库并结合数字基因表达(digital gene expression,DGE)对不同处理的黏虫进行测序,并运用实时荧光定量PCR(realtime quantitative polymerase chain reaction,RT-qPCR)技术验证12个P450基因的表达情况。结果表明,用于处理黏虫的氯虫苯甲酰胺和氟虫腈亚致死浓度LC_(10)分别为0.15、13.66 mg/L;对照样品、氟虫腈处理样品和氯虫苯甲酰胺处理样品分别获得59 521 504、64 838 148和41 722 990个原始序列数据,分别获得57 441 216、62 368 912和40 285 164个过滤后的序列数据;过滤后的序列长度分别为8.62、9.36和6.04 G;碱基错误率均为0.02%;Phred数值大于20、30的碱基占总碱基的百分比均高于90.59%;鸟嘌呤+胞嘧啶(guanine cytosine,GC)含量分别为47.16%、48.94%和47.55%,表明转录组测序质量较高;黏虫受氯虫苯甲酰胺胁迫后,29个P450基因表达量上调,27个P450基因表达量下调;黏虫受氟虫腈胁迫后,23个P450基因表达量上调,26个P450基因表达量下调;12个P450基因表达量的RT-qPCR技术检测结果与DGE测序文库显示的结果基本一致。