鉴于水源11类群近年来一直处于优势地位,为简化其检测手段,本研究利用RAPD技术对该类群的8个主要致病类型进行了多态性分析,以寻找其中主要流行类型的特异性分子标记。结果如下:共筛选出10个碱基随机引物190条,其中94条可得到稳定清晰的扩增图谱,用该94条引物进行RAPD分析,发现各致病类型间遗传变异丰富;以引物S1410扩增得到了水源11-4的特异性DNA条带;以引物S1412和S1304扩增得到了水源11-14的特异性DNA条带;对引物S1304扩增得到的特异性DNA条带回收、克隆和测序,设计了1对19bp/18bp的引物,并成功地将其转化为对水源11-14特异的SCAR标记。以上结果表明,通过规模筛选来寻找小麦条锈菌生理小种的特异性DNA片段,并将其转化为稳定的SCAR标记,有可能建立起中国小麦条锈菌流行生理小种的快速分子鉴定体系。

近几年来,山西红枣发生了1种严重的果实病害,症状表现为果顶或果肩部位形成红褐色的病斑。本研究以壶瓶枣为材料,对病菌进行分离。通过室内和田间致病性测定以及人工接种后再分离病菌,证明编号为CN535的真菌菌株为该病的致病菌。该病菌在PDA上7d菌落直径达69.2~73.5mm,基内菌丝和气生菌丝均发达,具明显的浅灰与墨绿色的同心轮纹;分生孢子单生或短链生,具纵横隔膜和短喙,大小为(22.5~40.0)μm×(8.0~13.5)μm,为典型的Alternaria属真菌特征。其rDNAITS序列分析结果表明该菌与A. alternata、A. tenuissima、A. longipes、A. mali和A. citri的同源性均为100%。用2对链格孢菌的专用引物AAF₂/AAR3和Aalt-F/Aalt-R分别扩增出相对应的341和450bp的片段。综合形态特征和分子分析结果,确定壶瓶枣褐斑病的病原菌为A. alternata (Fries) Keissler。

腐烂病是我国苹果树上一种严重的树干皮层腐烂病害,本研究拟探索快速、可靠、稳定及操作简便的病害室内评价方法。在"富士"苹果离体叶片、嫩梢、果实和枝条上造成不同伤口,强致病菌株03-8接种,25℃保湿培养后调查发现,不接菌对照有、无伤口均不发病;各种伤口接种病原菌均能发病,但伤口类型对病害影响很大。叶片正面较反面更有利于发病,叶片正面1针和10针的刺伤发病无明显差异;嫩梢叶痕接种较刺伤接种发病轻;果实表面针刺1针和10针及去除果皮造成伤口对发病影响大,差异显著;枝条烫伤接种后10d发病明显而其它材料在接种后1.5~2d即可发病。进一步用4个致病力不同的菌株验证评价方法的可靠性和稳定性,发现接种完全展开叶、嫩梢和枝条均可以充分显示不同菌株之间的致病力差异,但前两者重复性好、试验周期短、鉴定效率高,且操作简便、材料易得,因此建议用离体叶片或嫩梢作为材料,针刺1针接种,25℃保湿培养2d后调查,作为室内准确、快速评价苹果树腐烂病的方法,该方法可用于筛选抗病材料、评价分离株的致病性和药剂的防病效果等。

葡萄受卷叶伴随病毒侵染后,树势减弱,抗逆性变差,果穗着色不良,成熟期推迟,含糖量降低。目前已报道11种葡萄卷叶伴随病毒(Grapevine leafroll-associated virus,GLRaV)。为提高检测效率,降低检测费用,本文在研究单个卷叶伴随病毒RT-PCR检测技术基础上,对4种葡萄卷叶伴随病毒的多重RT-PCR模板浓度、引物浓度和退火温度进行优化,建立了同时检测葡萄卷叶伴随病毒-1(GLRaV-1)、葡萄卷叶伴随病毒-3(GLRaV-3)、葡萄卷叶伴随病毒-4(GLRaV-4)和葡萄卷叶伴随病毒-5(GLRaV-5)的多重RT-PCR技术体系。模板浓度、引物浓度、Taq DNA聚合酶浓度、退火温度和循环次数对多重RT-PCR检测结果均有较大影响,而在一定范围内改变延伸时间和dNTP浓度对检测结果影响较小。对4种葡萄卷叶伴随病毒的PCR产物进行克隆和测序,扩增基因片段与GenBank中登录的基因序列同源性为95%~99%。所建立的多重RT-PCR技术检测田间样品效果良好。

根据5种病毒小西葫芦黄花叶病毒(Zucchini yellow mosaic virus,ZYMV)、西瓜花叶病毒(Watermelon mosaic virus,WMV)、烟草花叶病毒(Tobacco mosaic virus,TMV)、南瓜花叶病毒(Squash mosaic virus,SqMV)和黄瓜花叶病毒(Cucumber mosaic virus,CMV)的核苷酸保守区序列,设计特异性引物对,从影响多重RT-PCR (mRT-PCR)扩增的引物浓度、Mg²⁺浓度、Taq DNA聚合酶浓度、dNTPs浓度、退火温度等方面进行反应体系的优化,建立了一种能够同时检测ZYMV、WMV、TMV、SqMV和CMV的多重RT-PCR技术体系,并进行了实际应用。在一个体系中对上述5种病毒复合侵染的西瓜材料进行多重RT-PCR扩增,得到与试验设计相符的5条特异性条带,依次是542、485、410、354和293bp。该体系实现了对侵染西瓜的5种病毒的同时检测,极大地提高了检测效率,降低了检测成本,体现了多重RT-PCR的优越性。

为了阐明水稻白叶枯病菌(Xanthomonas oryzae pv. oryzae,简称Xoo)鞭毛基体组分蛋白基因fliExoo的生物学功能,本研究用Gm^R抗性基因标记交换法成功构建了基因缺失突变体△fliExoo。与野生型菌株PXO99^A相比,△fliExoo鞭毛缺失,菌体易沉降,在0.3%半固体培养基上运动能力明显降低;尽管生长速率和胞外纤维素酶活性无明显变化,但胞外多糖产生和生物膜形成能力明显下降;对水稻品种日本晴的致病性和对非寄主烟草的致敏性反应明显减弱。基因互补可以使上述突变表型恢复。因此,鞭毛基因fliExoo突变不仅影响了细菌鞭毛依赖的运动性,而且对病原菌毒性相关的表型也产生了影响。

以海口香蕉束顶病株的幼嫩假茎和叶片总DNA为模板,通过反向PCR法克隆了香蕉束顶病毒(Banana bunchy top virus,BBTV)海口分离物(命名为BBTV-HaiKou)的6个DNA组分的全长序列。结果表明,DNA1~6序列全长分别为1106、1040、1058、1040、1013和1082nt。各组分非编码区各包含一个茎环共同区和一个主要共同区,同源性分别为91.55%和88.45%。各组分编码区均编码一个开放可读框(ORF),其中DNA1 ORF内部还有一个小的ORF。序列分析表明,BBTV-HaiKou分离物与其它分离物间的DNA1最为保守,DNA2变异最大。DNA1组分核苷酸序列与亚洲组、南太平洋组各分离物及ABTV (Abacá bunchy top virus)分离物同源性分别为93.1%~99.1%,89.6%~90.7%和76.2%~77.4%,相应的编码蛋白氨基酸序列同源性在BBTV和ABTV中分别为93.4%~100%和85.7%。根据Karan等的分类方法,确定BBTV-HaiKou分离物属于亚洲组成员。

本研究表明欧洲小麦品种Mega对我国小麦条锈病重要流行小种CYR30、CYR31、CYR32、Su-4和Su-14在苗期都具有良好的抗病性。采用小麦条锈菌小种CYR30对Mega与感病小麦品种铭贤169杂交的F₁、F₂和BC₁代及双亲进行苗期抗病性遗传分析,结果表明,Mega对CYR30的抗性由1对显性基因独立控制。采用SSR标记技术对其携带的抗性基因进行分子标记,在237对SSR引物中,发现位于5BL上的2个SSR引物位点Barc232、Wmc640在双亲和抗、感池间能扩增出稳定的特异性片段,与抗病基因连锁的遗传距离分别是3.7cM和8.6cM,暂命名为YrMe。本研究结果为科学利用Mega抗条锈基因培育抗病品种提供了依据。

根据已克隆的马铃薯Y病毒坏死株系(PVY^N)的基因组序列设计引物,利用PCR扩增HC-Pro、CI、NIb和CP4个基因的3'端400bp cDNA区段,分别反向插入含有査尔酮合成酶基因(Chalcone synthase,CHS)内含子的双元载体pRCHS中,构建了含内含子的发夹结构RNA (intron splicing hpRNA,ihpRNA)表达载体pRCHS-HC-Pro、pRCHS-CI、pRCHS-NIb和pRCHS-CP。利用农杆菌介导法转化烟草品种NC89,获得了多种转基因植株。病毒抗性检测的结果显示:转pRCHS-HC-Pro、pRCHS-CI、pRCHS-NIb和pRCHS-CP的烟草中,抗性植株的比例分别为55.34%、73.69%、61.54%和84.21%。大分子RNA和siRNA的Northern blot分析表明,目的片段转录产物的积累量与转基因植株的抗病性呈负相关,转基因植株中可检测到siRNA,说明所获得的抗病性是RNA沉默介导的。

在马铃薯环腐病区采集健康的马铃薯Solanum tuberosum块茎,从中分离到1株对马铃薯环腐病菌Clavibacter michiganense subsp. sepedonicum具有强拮抗作用的内生细菌P1。经形态观察、生理生化鉴定,菌株P1归属为Bacillus sp.,进一步经16SrDNA序列对比分析,确定为巨大芽孢杆菌B. megatherium。经证明,P1菌株的培养液抗菌粗提物属于蛋白类,该抗菌蛋白对紫外光不敏感,pH为7.0时抑菌活性最强,温度高于80℃时抑菌活性明显下降。温室试验表明,P1菌株能显著提高马铃薯植株的株高、茎粗、产量及大薯率,其对马铃薯环腐病防效达53.4%。

本研究从菌株内生定殖、促生、抑制和诱导防御酶活性4方面探讨了红树内生细菌RS261菌株防治辣椒疫病的机理。结果表明,该菌株能进入辣椒等多种陆生植物体内定殖,在辣椒体内的定殖时间达26d以上,且对辣椒生长表现有明显的促进作用;抑制作用测定发现,RS261能够分泌产生抗菌物质抑制辣椒疫霉菌丝的生长和游动孢子囊的形成;辣椒体内丙二醛(MDA)含量及防御性酶活测定结果显示,经过RS261培养液处理后,辣椒苗体内丙二醛(MDA)含量和超氧化歧化酶(SOD)、过氧化物酶(POD)以及过氧化氢酶(CAT)的活性等均较仅接种疫霉病菌的处理低,但可显著诱导辣椒体内苯丙氨酸解氨酶(PAL)的活性。

保护地连作是根结线虫为害严重的主要原因,为评估连作年限对根结线虫数量及其与土壤线虫数量关系,选择典型的不同连作时间的保护地(种植番茄),研究番茄生长期间根围土壤不同土层的根结线虫二龄幼虫(second-stage juveniles,J2)与自由生活线虫数量的动态变化。结果表明:根结线虫J2数量随连作年限增加而增多,0和5-year土壤中根结线虫数量少,平均根结线虫数为1.1和2.1条/100g干土,8、10和12-year土壤中根结线虫数量平均值分别为154.9、68.3和861.8条/100g干土;在0~30cm土层,根结线虫J2数量随土层加深而增加,主要分布在20~30cm土层。土壤自由生活线虫数量随土层的增加而增多,连作对自由生活线虫数量无显著影响。0和5-year土壤中根结线虫群体占土壤线虫总数的比例小,属稀有类群,在土壤线虫中处于稳定的群落结构状态。连作8、10和12-year的土壤中根结线虫J2为优势类群,土壤中自由生活线虫数量与根结线虫数量呈现相反的趋势,根结线虫J2占土壤线虫总数的比例增加,自由生活线虫与根结线虫J2数量的比值显著降低(P<0.05)。

Soft rot disease often affects Phalaenopsis amabilis during the growing season. However, the pathogen of the disease is remaining poorly studied. In this study, bacterial strain R1 was isolated from soft rot tissues in Wuhan. The pathogenic, morphological, physiological, biochemical tests and 16S rDNA sequence analysis were carried out. The homology of 16S rDNA sequence between strain R1 and Pseudomonas grimontii was 99.72%, and its physiological and biochemical properties were also similar to those of Pseudomonas grimontiis. All these evidences indicated that strain R1 could be identified as a novel strain of Pseudomonas grimontii. The pathogenicity of the novel isolate was proved according to the Koch's postulates. This is the first report that Pseudomonas grimontii can cause soft rot disease of Phalaenopsis amabilis.

A multiplex reverse transcription polymerase chain reaction (multiplex RT-PCR) method was developed for the simultaneous detection and discrimination of three sweet potato potyviruses:Sweet potato feathery mottle virus (SPFMV), Sweet potato latent virus (SPLV) and Sweet potato virus G (SPVG). Three compatible sets of primers specific for each virus were designed in conserved regions of the coat protein (CP) gene for use in multiplex RT-PCR assay, and producing three distinct fragments 300, 420, and 600 bp, indicating the presence of SPFMV, SPLV and SPVG respectively. The individual RT-PCR assays and the multiplex assay were optimized for highest sensitivity and specificity. This study fulfilled the need for rapid and specific sweet potato potyvirus diagnostic tool and that also had the potential for investigating the epidemiology of sweet viral diseases.

The activity change of defensive enzymes and PR-1a gene expression of tobacco (Nicotiana tabacum) seedling induced by chito-oligosaccharides were studied. The results showed that high level systemic acquired resistance (SAR) was expressed in tobacco plants treated with chito-oligosaccharides solution at the concentration of 50 μg/mL. PAL activity increased greatly with 2 peaks, the activity of SOD decreased initially followed by an increase with higher increment, and the activity of POD peaked early followed by a gentle fall in chito-oligosaccharide treated plants. The PR-1a gene was strongly expressed in tobacco due to systemic acquired resistance induced by chito-oligosaccharides. At 168 h after inoculation the expression quantity (co-pies/2 μL) of PR-1a gene was increased to 2 469.6 in treated tobacco leaf, reached 392.6% than that at 0 h after inoculation, it was increased 3.05 times of that in untreated control.

The growth and sporulation of Clonostachys rosea strain 67-1 in PD broth was observed and figured out. A large amount of submerged spores were obtained in shake flask with proprietary Czapek me-dium. Meanwhile, the colonies of strain 67-1were cultured in PDA plate and aerial spores were collected by elution. Resistances of submerged spores and aerial spores to high temperature, dry condition and UV treatment were determined. The results showed that the survival of the submerged spores kept in 60℃ for 30 min was 76.7%, while the aerial spores were hardly germinated in the same condition. After two weeks' drying treatment, the spore activities were 89.2% and 29.3%, respectively. Similarly, the activity of submerged spores was 72.6% and that of aerial spores was 19.7% when exposing to UV for 1 min. It is illuminated that deep submerged fermentation is more efficient than solid culture for strain 67-1 to produce chlamydospores, which act as main component in biopesticide mass production.

The sensitivity of 113 isolates of wheat powdery mildew (Blumeria graminis f. sp. tritici) sampled from 6 provinces or cities in 2008 to temperature was tested by detached leaf segment method with setting up 5 different temperatures indoor. The results showed the mean ET₅₀ (which represents the temperature that is required to obtain 50% of the maximum effect) of all isolates tested was 23.02℃. ET₅₀ values of 17.70% isolates were more than 24℃. The highest and the lowest ET₅₀ of isolates were 25.22℃ and 19.42℃, respectively. There were a certain differences for isolates sensitivity to temperature among different provinces or cites. It was also found that when temperature increased during 22-26℃, the latent period of isolates prolonged, and the latent period of different isolates was different at the same temperature, too. These results will provide a reference for the oversummering division of wheat powdery mildew, as well as the effect of climate to the disease.

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。

以大白菜抗病品种秦白2号和感病品种06J31为材料,研究其接种黑腐病菌种YL-17后保护酶活性、超氧阴离子和MDA含量的变化。结果显示:接种后不同抗性的材料总体上表现为超氧阴离子(O₂^-_·)含量先降后升,SOD活性逐步降低,POD活性MDA含量逐步升高,CAT活性则二者变化不一致(抗性材料表现为升-降-升,感性材料表现为先降后升)。由此表明:黑腐病菌侵染大白菜后,抗病材料与感病材料相比,能迸发较多的O₂^-_· 并刺激抗氧化物酶SOD和POD活性升高,使MDA含量处于较低的水平,有利于提高秦白2号抗黑腐病和抗氧化能力。

利用RT-PCR技术扩增得到了葡萄卷叶伴随病毒-3(Grapevine leafroll associated virus-3,GLRaV-3)中国分离物的外壳蛋白(coat protein,cp)基因。序列分析结果表明,cp基因的长度为942bp,与已报道的其它GLRaV-3分离物的cp核苷酸相似性为91%~99%,编码的氨基酸相似性为95%~100%。将此基因克隆到原核表达载体pET-28a (+)上,转化大肠杆菌BL21(DE3) plysS后用终浓度为1mmol/L的IPTG进行诱导表达,SDS-PAGE及Western blotting分析表明,cp在大肠杆菌中可表达出分子量约为35kDa的蛋白。纯化表达产物后免疫家兔制备抗血清。A蛋白酶联免疫吸附测定(PAS-ELISA)及斑点免疫结合测定(DBIA)结果显示,制备的特异抗血清可用于检测田间感病葡萄样品中的GLRaV-3。

以玉米自交系黄早四和黄早四Ht2近等基因型系为材料,利用cDNA-AFLP差异显示方法,分析玉米抗大斑病Ht2相关基因受大斑病菌1号小种胁迫后的差异表达情况。用筛选的70对引物在黄早四Ht2中扩增出76个差异显示片段,对差异条带回收、克隆和测序,并在NCBI上进行BLAST分析,有52个在GenBank中发现相似性较高、功能已知的EST序列,按其功能分为以下8类:基础能量代谢、跨膜运输蛋白、抗逆或抗病相关蛋白、蛋白质代谢、染色体组成蛋白及DNA复制和转录中的蛋白、信号传导、生长发育调控因子和转录因子,而其中以参与基础能量代谢的基因和抗逆或抗病相关基因所占比例较大。功能已知的52个差异表达片段在玉米1~9号染色体上均有分布,且在非亲和互作前期(6h)主要是一些信号相关基因的表达,12h表达的基因多属于抗病反应初期阶段的相关基因,与防卫有关的基因主要在48~72h表达,生长发育类基因在所检测的非亲和互作的不同阶段均表现作用,在非亲和互作后期主要是蛋白质代谢基因的表达。

Pseudomonas fluorescens 2P24是分离自山东小麦全蚀病自然衰退土的1株生物防治菌株,产生抗生素2,4-二乙酰基间苯三酚(2,4-diacetylphloroglucinol;2,4-DAPG)是其主要防病机制。2,4-DAPG是由phlACBD基因簇合成,受多种调控因子调控。本研究用Tn5转座子插入技术,获得1株phlA基因转录增强的突变体,其突变基因为抗生素合成的负调控基因phlF。与野生菌相比,phlF基因的缺失突变体中phlA的转录增强约100倍,抗生素产量提高492倍。同时,菌株2P24的phlF缺失突变体对病原真菌的拮抗作用明显增强。但2,4-DAPG过量表达菌株对多种作物种子根生长有抑制作用。

本文以抗病品种宜香2292(Oryzas ativa L. ssp. indica cv. Yixiang 2292)为材料,采用高效液相色谱技术(HPLC)和Real-time PCR技术研究了水稻矮缩病毒(Rice dwarf virus,RDV)胁迫下水稻内源赤霉素(GA₃)、生长素(IAA)和脱落酸(ABA)的动态变化。结果表明,受RDV侵染后,病株体内GA₃含量显著低于健株,在显症后的第1d和第10d最为明显,分别较健株低6.28和5.92倍;IAA含量呈现波动变化,但病株体内的IAA含量始终较健株低,在显症后的10d最为明显,比健株低3.58倍;与GA₃和IAA相反,病株体内ABA的含量始终高于健株,显症后的第1d和第13d最为明显,分别较健株高2.29和2.84倍。Real-time PCR定量检测了植物内源激素相关基因mRNA的表达,结果显示,GA₃代谢相关的氧化还原酶基因表现为下调,而IAA和ABA代谢相关的Cullin-1和P-glycoprotein1基因表现出不同程度的上调。以上结果表明:水稻矮缩病的症状表现可能与病株体内的植物内源激素失调有关。

烟草曲茎病毒(Tobacco curly shoot virus,TbCSV) Y35分离物(Y35)和中国番茄黄化曲叶病毒(Tomato yellow leaf curl China virus,TYLCCNV) Y10分离物(Y10)是从云南烟草上分离的2种单组份双生病毒,分别伴随卫星DNAβ(Y35β及Y10β)分子。将Y35和Y10分别伴随其同源或异源卫星(Y35β或Y10β)接种本氏烟和心叶烟,症状观察表明,无论Y10还是Y35伴随异源卫星DNAβ时引起的症状均与其伴随同源DNAβ引起的症状明显不同,且Y10β伴随Y35致病性最强。Southern印迹结果表明,Y10β及Y35β均可被异源辅助病毒稳定复制,但其复制水平明显低于伴随同源病毒时的水平,且植株中病毒和卫星的积累量并不完全与症状严重程度直接相关。

类FMRF酰胺多肽(FMRFamide-like peptides,FLPs)在控制寄生性线虫侵染、取食和繁殖过程中起着重要作用。在防治线虫时,FLPs信号系统经常成为药物的作用靶标。本研究以香蕉相似穿孔线虫(Radopholus similis)为材料,用RT-PCR和RACE方法,获得了类FMRF酰胺多肽flp-14基因的cDNA全长序列,命名为Rs-flp-14。此基因cDNA序列全长为569bp,包括一个357bp的开放阅读框(0RF),编码含119个氨基酸的蛋白,分子量与等电点分别为12.85KD和9.41。序列分析表明Rs-flp-14编码2个FLP肽(2xKHEYLRFG),N端具有27个氨基酸残基组成的信号肽。Southern杂交显示其为单拷贝基因。cDNA与基因组DNA重叠分析表明基因包含三个内含子,四个外显子。聚类分析表明该基因与猪蛔虫的As-flp-14同源,相似度最高(64%)。原位杂交结果显示Rs-flp-14基因表达位置在线虫的神经环部位。本研究结果将为研究该基因功能奠定基础,并为以FLPs为作用靶标的防治线虫药物提供理论依据。

筛选能显著诱导马铃薯块茎抗晚疫病的微生物源激发子,并探讨其诱导抗病性机理。分别将31种微生物的发酵液(F-0)以及其中真菌和放线菌的胞内提取物(F-1)、胞壁提取物(F-2)单独或复配后诱导处理马铃薯块茎切片,对其中诱导抗病效果显著的激发子进一步分析其中的有效诱抗物质及诱导处理后的生理变化。获得了9种较好的激发子,诱抗效果均达50%以上,其中放线菌A5295发酵液型激发子诱抗效果最高,达63.97%,且单一激发子复配后诱抗活性更高,其中MK与A5295的复配型激发子诱抗效果最好,达66.67%。MK和A32910b发酵液中有效诱抗物质均为饱和硫酸铵沉淀(A组分)、以及硫酸铵沉淀后再用乙醇沉淀(B组分)所得的物质。诱导处理后块茎中POD、PAL、PPO活性均明显高于对照,且在抗性表现出之前迅速增加达到峰值。此外,块茎中可溶性蛋白含量比对照增加41.53%,并有一些新蛋白产生。结果表明复配激发子比单一激发子诱抗活性更高,激发子中有效诱抗物质均为A和B组分,诱导的抗病性可能与块茎中POD、PAL、PPO活性增高,可溶性蛋白含量增加以及新蛋白质的产生有关。

用感细菌性条斑病品种9311为母本,4个普通野生稻抗源DY3、DY16、DY17、DY20为父本,组配了9311/DY3、9311/DY16、9311/DY17和9311/DY204个组合的F₁、F₂和B₁C₁群体。在水稻分蘖期,用广西细菌性条斑病菌优势致病型菌株JZ28,以针刺法对各世代进行接种鉴定和遗传分析。结果表明,对DY3,F₂群体抗感分离比为39:544,卡方测验x²=0.1245 < x_0.05,1²,符合1R:15S的理论比例;对DY16,F₂群体抗感分离比为94:343,卡方测验x²=2.655 < x_0.05,1²,符合1R:3S的理论比例;对DY17,F₂群体抗感分离比为41:501,卡方测验x²=1.382 < x_0.05,1²,符合1R:15S的理论比例;对DY20,F₂群体抗感分离比为43:489,卡方测验x²=2.745 < x_0.05,1²,符合1R:15S的理论比例。结合各个组合F₁和B₁C₁植株对细菌性条斑病表现为感至高感,据此推知DY3、DY17、DY20的抗性由2对隐性抗性基因控制;DY16的抗性由1对隐性抗性基因控制。

本研究应用简单序列重复区间(inter-simple sequence repeats,ISSR)标记技术对福建省10个县市100个水稻纹枯病菌菌株的遗传多样性进行了分析。从16个随机引物中筛选出3个重复性好、特异性高的引物对菌株进行ISSR-PCR扩增,共产生41个ISSR分子标记,其中90.2%的片段具有多态性。PopGene分析结果表明,种群的平均多态位点百分率为55.14%,Shannon表型多样性指数I平均为0.2953,具有较高的遗传多样性;种群间存在着明显的遗传分化(Nei's信息多样性指数h平均值为0.1991,G_st平均值为0.6423),聚类分析可将它们分成5个遗传聚类群。同时还进行了菌株生长速率和致病力的测定。ISSR遗传聚类组群的划分与菌株的地理来源有明显的相关性,但与菌株的致病力和生长速率都没有明显的相关性。以上结果为进一步开展水稻纹枯病菌群体遗传分析和纹枯病防治提供了一定的理论依据。

本文研究了万寿菊根提取物中西瓜枯萎病菌的抑菌活性成分及其抑菌作用机理。结果表明,在4类主要化学物质中,精油类含量最高(59.8%),且抑菌效果最佳;10mg/mL精油提取物的抑制率在24、36和48h分别为62.83%,58.31%和56.30%。在西瓜枯萎病的3个主要发病期(苗期、伸蔓开花期和座果期)施用精油提取液,能有效抑制西瓜枯萎病菌,促进植株生长,同时提高西瓜植株的POD和SOD酶活性,并维持CAT活性,有效减轻了西瓜枯萎病菌对植株的毒害作用。

为了鉴定解淀粉芽孢杆菌YN-1发酵液中抑制植物病原真菌物质的主要种类及其相对含量,本研究根据已知的脂肽抗生素合成相关基因序列设计了4对特异引物对YN-1菌株进行了检测,对PCR产物克隆、测序,然后采用酸沉淀法从菌株发酵液制备出抑菌物质粗提液,平板拮抗试验确定其抑菌活性,最后对活性粗提物进行HPLC-ESI-MS和MALDI-TOF-MS分析。3对引物的扩增产物经克隆、测序和BLAST分析,表明解淀粉芽孢杆菌YN-1基因组中含有sfp、fenB、ituA或bamA基因。YN-1菌株发酵液的粗提物对棉花枯萎病菌具有抑菌活性,质谱分析发现活性粗提物中含有C14-Iturin A、C15-Iturin A、C16-Iturin A、C14-Fengycin A、C15-Fengycin A、C16-Fengycin A、C17-Fengycin A、C16-Fengycin B和C17-Fengycin B9种脂肽抗生素,其中C16-Iturin A、C14-Fengycin A、C16-Fengycin A、C17-Fengycin A和C17-Fengycin B5种脂肽抗生素为活性粗提物中的主要成分。该研究结果为利用YN-1菌株开发成微生物杀菌剂奠定了基础。

Thielaviopsis basicola is a soil-borne plant pathogen which causes root rot disease in tobacco plants. Detection and monitoring of T. basicolain soil is of great significance to control this disease. Based on the differences in internal transcribed spacer (ITS) sequences of T. basicola and other fungal pathogens, a specific primer pair Tb1/Tb2 for T. basicolawas developed. The results showed that the primer pair gave a single amplicon of 330 bp from T. basicola and revealed no undesirable cross-reaction with other seven soil-borne pathogen isolates and three tobacco rhizosphere dominant fungi isolates. With a series of 10-fold genomic DNA dilutions of T. basicola, the detection limit of 1 pg/μL in conventional PCRand100 fg/μL in real-time quantitative PCR was achieved. With DNA from the soil inoculated with different numbers of T. basicola conidia, the detection limit was 10 conidia per reaction in conventional PCR and 0.4 conidia per reaction in real-time quantitative PCR.

The rDNA-IGS of 11 Ustilaginoidea virens isolates from different geographical regions in China were amplified by PCR and sequenced. The results showed that the rDNA-IGS fragments consisted of one cen-tral variable region and two lateral conservative regions. The two conservative sequences among the11 isolates shared the sequence similarity of 99.44%. The variable region consisted of 2, 4, or 6 of 77 bp-length-repeats in the isolates and the sequences of the repeat units were high conservative. Primers UIGS Ⅲ and UIGS IV were designed for amplification of the variable region in the rDNA-IGS.

The damage and symptoms of jujube fruit soft rot in Xinzheng, Henan Province, were investigated from 2006 to 2008, and the pathogen was identified based on the morphological characteristics and the ITS se-quence of ribosome DNA. The results showed that the aerial mycelium of colony was white in color in the first four days, then turned gray after incubation on PDA for 5-6 d at 25℃ and became black two weeks later. The mycelia grew luxuriantly with velvet character. The pycnidium was flask-shaped with a height of 196.9μm and a width of 213.3μm on the avereage. The conidium was colorless with single cell and had the shape of spindle, its size was (15.0-20.0)μm× (4.5-6.5)μm. The conidiophore was fastigiate. The homology of ITS sequence of ribosome DNA between the tested strain NXK and Botryosphaeria dothidea (GenBank ac-cession number:AJ938005) reached 99.87%, with a difference of only two base pairs. Based on the results of both morphological characters and molecular identification, the pathogen of jujube fruit soft rot in Xinzheng was identified as B. dothidea (Moug. ex Fr.) Ces. et de Not..

The effect of silica nano-particles, tetraethyl orthosilicate (TEOS) and sodium silicate in solution culture on wheat powdery mildew and its action mechanism to pathogen attack were studied. The results showed that wheat seedlings cultured in nutrient solution with silicon compounds alleviated wheat powdery mil-dew, the control efficacies of TEOS and sodium silicate treatments were 54.08% and 51.36%, respectively, but that of silica nano-particles treatment was only 1.02%. Scanning electron micrograph and X-ray energy dispersive analysis (SEM-EDX) showed that wheat leaves cultured in nutrient solution with TEOS and sodium silicate had more silicon deposition than that of silica nano-particles. It indicated that wheat possessed the equal good imbibe ability to TEOS and sodium silicate, but poor to silicate nano-particles. When wheat leaves were infected with Blumeria graminis f. sp tritici, silicon might has a physical prohibition effect to pathogen spore penetration.

根结线虫是世界农业生产中危害最大的植物病原之一,目前仍缺乏安全有效的防治措施。深入揭示寄生线虫与植物之间互作的分子机制,利用生物技术进行抗性育种被认为是最有前景的抗线虫策略。在根结线虫基因组学研究方面,目前已经构建了北方根结线虫AFLP遗传连锁图谱,南方根结线虫和北方根结线虫基因组测序也已完成;基因组的注释和比较基因组学分析,较全面地描述了根结线虫的遗传组成;以差异表达分析和比较基因组学为主的方法鉴定了大量的重要基因;以RNA干扰、植物转化和蛋白互作为主的根结线虫基因功能研究也取得了一些进展。本文就根结线虫基因组学研究予以综述,并进一步探讨其研究方向和可持续抗线虫新策略的发展前景。

兰花褐斑病菌(Acidovorax avenae subsp. cattleyae)是兰花上一种重要进境检疫性有害生物,可通过植株和种子远距离传播,其传染性强,对兰花危害性大。根据核糖体ITS序列设计了TaqMan-MGB探针并建立了实时荧光PCR检测方法。该方法能够特异性检测兰花褐斑病菌,所有供试的目标菌株检测结果均为阳性,而其它28个对照菌株(含同属内其它种及avenae种下其它亚种)均为阴性。利用该方法从发病兰花植株总DNA中检测到该病菌。本方法灵敏度高,检测极限达9×10^-9μg DNA,操作方便快速,结果可靠,适合于口岸兰花的进出境检疫及兰花种苗健康质量控制。

罗城仫佬族自治县是广西毛葡萄主产区。穗轴褐腐病是近年为害毛葡萄花穗穗轴的主要病害。作者根据病原菌形态、致病性,并利用分子鉴定方法,将该病的致病菌鉴定为可可毛色二孢(Lasiodiplodia theobromae)。该病菌生长最适温度为28~30℃;可生长pH值为3~11.5,其中,pH 3~4利于病原菌产生分生孢子;能有效利用蔗糖、葡萄糖、可溶性淀粉等14种供试碳源,但以葡萄糖最好;能利用DL-丙氨酸、硝酸钠、DL-甲硫氨酸等10种供试氮源,但以DL-丙氨酸最适。

利用DSN (duplex-specific nuclease)均一化与SMART (switching mechanism at 5' end of RNA transcript)技术构建玉米弯孢霉叶斑病菌菌丝的均一化全长cDNA文库。经检测原始文库的滴度为1.4×10⁶ pfu/mL,重组率为96.9%,插入片段平均长度大于1.0 kb。从文库中随机挑取192个cDNA克隆测序,获得173个高质量的EST序列,其中有5个跨叠群(conting),168个独立克隆,全长cDNA克隆占57%。BLASTx分析表明,有18.5% EST与GenBank中无任何同源性,有81.5% EST与GenBank中报道的功能已知或未知蛋白具有相似性。

为了探讨寡糖诱导物的分子结构与诱导抗病性之间的相互关系,以7个化学合成的寡糖为诱导物,研究了烟草植株对黑胫病的诱导抗性。结果表明,β-1,3-乙酰氨基葡聚四糖、β-1,4-乙酰氨基葡聚三糖和β-1,4-乙酰氨基葡聚四糖的诱导处理对烟草黑胫病表现活体抗性,相对诱导效果分别为62.5%,50.0%和75.0%;β-1,3-乙酰氨基葡聚四糖、β-1,4-乙酰氨基葡聚三糖和β-1,4-乙酰氨基葡聚四糖的诱导处理对烟草黑胫病表现离体抗性,相对诱导效果分别为56.25%,50.0%和62.5%。研究结果表明寡糖的聚合度以及寡糖主链的糖苷键连接方式可能是影响诱导抗病性的重要因子,不同浓度寡糖处理后烟草的诱导抗病性有差异。

为探讨活性氧和细胞质钙离子在小麦抗条锈病反应中的作用,以小麦品种洛夫林13与具有不同致病力的单孢锈菌CY29、CY25的互作体系为平台,对锈菌侵染后小麦叶片中活性氧(ROS)积累、保护酶系(SOD、CAT和APX)活性变化动态、细胞质膜的透性改变及细胞质钙离子浓度变化做了研究。结果表明,不亲和锈菌CY25侵染可引起小麦叶片内2次ROS的爆发,第1次出现在接种后前期(接种后第2天),强度较小,第2次出现在接种后期(接种后第5天),强度较大;亲和锈菌CY29侵染只引起1次ROS爆发,出现在接种后期(接种后第5和第6天之间),但强度极大。过敏性坏死反应HR只出现在不亲和互作小麦叶片上前期ROS爆发之后,表明前期ROS爆发与HR的产生有关。伴随着后期小麦叶片中强度极高的ROS的爆发,叶片细胞原生质膜遭到了破坏,细胞内物质外渗,细胞不久便死亡,表明高强度的ROS爆发会导致细胞死亡。根据不同互作体系ROS爆发时期SOD、CAT和APX等保护酶的活性变化分析,不亲和互作体系前期强度较小的ROS爆发主要成分是H₂O₂,后期强度较高的ROS爆发主要成分是O₂^-_·和H₂O₂;亲和互作体系强度极高的ROS爆发主要成分是O₂^-_·和H₂O₂。由此说明H₂O₂是引起小麦抗病反应HR发生的因素,而O₂^-_·则是引起细胞死亡的因素。细胞质钙离子浓度变化研究表明,HR的发生与细胞质钙离子浓度增加相关。细胞质钙离子浓度的降低推迟了HR的发生,这说明小麦叶片细胞内细胞质钙离子浓度的增加是HR的必要条件,同时也说明Ca²⁺是植物HR的胞内第二信使参与植物抗病防卫反应。