2016年北京地区玉蜀黍黑粉菌遗传多样性分析

 2016年从北京市10个区采集317份玉米瘤黑粉病样品,选择核糖体RNA内转录间隔区的ITS rDNA和28S rDNA基因作为持家基因,通过单核苷酸多态性技术(Single Nucleotide Polymorphism,SNP)分析北京市10个区玉蜀黍黑粉菌的遗传多样性。ITS rDNA基因分析表明,有3个菌株的序列存在1个颠换突变位点,产生两种单倍型,采用邻接法构建的系统发育树将317个菌株分为2个群体;28S rDNA基因分析表明,有54个菌株的序列存在1个颠换突变位点,产生两种单倍型,采用邻接法构建的系统发育树将317个菌株分为2个群体。中性检测表明北京市10个区的玉蜀黍黑粉菌符合中性进化模型。试验结果表明北京市10个区的玉蜀黍黑粉菌遗传多样性不高,遗传分化不明显。     

Comparative antifungal effect and mode of action of tetraconazole on Ustilago maydis

Tetraconazole, a new, recently introduced antifungal triazole, has been assayed in parallel with a number of standard analogues on various sensitive strains of Ustilago maydis. The values of EC₅₀ and EC₉₀ tetraconazole concentrations, determined on strain ATCC 14826 in agar, were 0.5 × 10⁻⁶ and 3.5 × 10⁻⁵ , respectively, in reasonable agreement with those needed to inhibit by 50% and 90%, respectively, the ergosterol biosynthesis in broth cultures. Squalene and 12 sterols have been extracted from the latter, characterized and quantified. Accumulation of 14α-methylsterols and reduction of ergosterol and other late precursors are consistent with the inhibition of 14α-demethylase caused by the title compound.     

基于CLIMEX模型的玉蜀黍霜指霉菌入侵风险分析

为明确我国禁止进境的植物检疫性有害生物——玉蜀黍霜指霉菌Peronosclerospora maydis在中国的适生性以及入侵风险,采用CLIMEX 2.0软件分析其适生性,用ArcGIS 10.2软件确定其适生范围和适生程度,并采用多指标综合评判法量化分析其入侵我国的风险等级。结果表明,玉蜀黍霜指霉菌在我国的适生范围主要分布在长江以南的西南山地丘陵玉米种植区和南方丘陵玉米种植区,高适生区主要分布在云南、贵州、四川、重庆、广东、广西、湖南、江西、福建、海南和台湾等省 （区、市）。玉蜀黍霜指霉菌作为专性寄生菌,可在玉米整个生长季侵染危害,经多指标综合评判法分析并计算得到其风险综合评价值R为2.12,表明该病菌入侵我国的风险等级属高度危险,一旦入侵,势必对粮食安全、农业安全和生态安全带来巨大威胁,建议加强检疫监测,严防该病菌入侵。     

禾谷镰孢荧光定量检测体系的建立及其在玉米苗枯病上的应用

为实现对田间土壤中禾谷镰孢Fusarium graminearum的定量检测,本研究构建了土壤含菌量与玉米苗枯病病情指数的回归模型。基于禾谷镰孢甾醇14α-去甲基化酶基因CYP51C序列,设计特异性引物HQ1-F/HQ1-R,利用引物建立实时荧光定量PCR(RT-q PCR)体系,选取4个玉米自交系品种进行室内苗枯病接种试验,调查其病情指数,利用RT-qPCR体系检测土壤禾谷镰孢含菌量,并对病情指数和土壤禾谷镰孢含菌量进行回归。结果表明,仅禾谷镰孢扩增出目的条带并且可从多种病原菌土壤中检测出。RT-qPCR的熔解曲线具有单一吸收峰,扩增曲线的循环阈值与模板浓度呈良好的线性关系,扩增效率为104.7%,标准曲线为y=-3.2137x+34.9560(R~2=0.9968),最低可检测到1 pg/μL的DNA。随着土壤禾谷镰孢接菌量的增加,单位土壤禾谷镰孢含菌量呈线性增加,即y=13.603x-85.370(R~2=0.9998)。4个玉米品种的病情指数与土壤禾谷镰孢含菌量的回归曲线分别为y=0.0789x+22.0590(R~2=0.7949)、y=0.0304x+7.8686(R~2=0.9579)、y=0.0458x+23.7540(R~2=0.5420)、y=0.0471x+32.0760(R~2=0.6753)。     

葡萄拟尾孢菌PCR检测方法的建立

<正>葡萄褐斑病又称叶斑病,是葡萄生产上的一种常见病害,根据病斑大小和病原物不同分为大褐斑病和小褐斑病。我国各地报道的葡萄褐斑病主要以大褐斑病为主,其病原为葡萄拟尾孢菌Pseudocercospora vitis(梁春浩等,2009),主要为害葡萄叶片,可造成叶片早期脱落,光合作用下降。目前该病的鉴别主要通过技术人员实地观察,主观性强,传统的病原分离培养和鉴定虽然结果准确,但需较     

玉米小斑病菌中一种维多利亚病毒的分离及其基因组全序列分析

 从玉米小斑病菌(Bipolaris maydis)中检测发现一种dsRNA (double-stranded RNA)病毒,暂命名为Bipolaris maydis victorivirus 2 (BmV2)。电子显微镜下观察到病毒粒子为二十面体球状,直径为40 nm左右、无包膜;病毒基因组为单条dsRNA核酸分子,长度为5 222 bp,其基因组结构与其他维多利亚病毒相似,包含两个开放阅读框(ORFs)。序列BLASTx分析发现,BmV2的基因组核酸序列与Coniothyrium minitans RNA virus Illinois isolate (CmRV-IL)具有较高的同源性(78.15%),后者CmRV-IL是单分体病毒科(Totiviridae)维多利亚病毒属(Victorivirus)的暂定种,两者的外壳蛋白(CP)和RNA依赖性RNA聚合酶(RdRp)的氨基酸序列之间的同源性分别为88.02%和89.87%,说明BmV2、CmRV-IL是同一种病毒的不同分离物。基于BmV2和选择的单分体病毒的RdRp氨基酸序列的系统发育分析表明,BmV2与单分体病毒科维多利亚病毒属中的病毒形成了一个单系进化支。     

Hydrophobin gene expression in the maize pathogen Cochliobolus heterostrophus

Filamentous fungi produce hydrophobins, small proteins localized on the outer surface of their cell walls and involved in growth and development. Hydrophobin gene expression depends on nutrient availability, light, and the activity of conserved signal-transduction pathways. We found four hydrophobins, one class I and three class II family members, in the Cochliobolus heterostrophus genome with high homology to other ascomycete hydrophobins, which present a typical conserved array of cysteines. The expression profile of a selected gene from each class was determined in a series of signaling-deficient mutants. Loss of either of two mitogen-activated protein kinase (MAPK) genes, CHK1 and MPS1, led to decreased hydrophobin class I (CHYD1) gene expression. Mutants in both MAPK genes had easily wettable colonies, but decreased CHYD1 expression was not the sole explanation for this phenotype. A significant elevation of hydrophobin class II (CHYD3) gene expression was measured in the chk1 mutant, suggesting a complex role for MAPK in controlling the expression of these hydrophobins. Similar but less marked tendencies were observed in G-protein α and β subunit loss-of-function mutants; however these showed no alteration in colony hydrophobicity. Overexpression of CHYD1 in the wild-type background caused a change in colony morphology and a small but significant increase in aerial growth. Thus G-protein and MAPK signal transduction influence hydrophobin gene expression and colony hydrophobicity. The connection between colony hydrophobicity and expression of the hydrophobin genes CHYD1 and CHYD3, however, is not one-to-one, indicating that additional factors determine colony-surface properties. The approach of using hydrophobin-overexpression mutants to investigate their role may be generalized to other hydrophobins and small secreted proteins.     

利用gusA基因在水稻组织中示踪和定量水稻白叶枯病菌的方法

 水稻白叶枯病菌(Xanthomonas oryzae pv. oryzae, Xoo)从自然孔口水孔或者伤口处侵入水稻叶片,在维管束中定殖、繁殖和扩展,引起典型的叶枯症状。可视化这个动态的病程过程和快速定量水稻组织中细菌的群体是Xoo-水稻互作研究中亟待突破的技术难点。本研究在PXO99^A菌株中表达gusA基因,将其置于lacZ启动子、hrpX启动子和不含有(T₁)₄终止子的hrpX启动子下,构建了3个示踪菌株PXO99^A_GUSRBS、PXO99^A_GUSX和PXO99^A_GUSX-。水稻上毒性测定结果显示,这3个示踪菌株与野生型PXO99^A展现相同的毒性。利用示踪菌株,通过注射接种法,能够观察到Xoo在水稻维管束中定殖和扩展的动态变化;通过喷雾接种法,能够观察到Xoo从叶尖或者叶缘侵入水稻叶片引起发病的特性;发现PXO99^A_GUSX和PXO99^A_GUSX-比PXO99^A_GUSRBS能够更加灵敏地反映这些病程。同时发现,PXO99^A_GUSRBS的细菌数量与受其侵染的水稻组织的GUS活性显著正相关。本研究也评价了利用GUS活性测定法精确和快速地定量水稻组织中细菌群体的可行性。以上结果表明这套GUS系统是非常有效的,能够示踪病原菌的侵染过程和监测细菌在水稻组织中的群体数量,这将为Xoo-水稻互作研究提供有力的技术支持。     

柑橘黄龙病菌过氧化物还原酶基因CLasPrx的克隆及功能分析

为解析柑橘黄龙病菌亚洲种Candidatus Liberibacter asiaticus（CLas）逃逸活性氧伤害的机理,通过克隆其过氧化物还原酶（peroxiredoxin,Prx）编码基因的全长序列,对CLasPrx蛋白序列进行生物信息学分析、多重比对及系统发育分析,采用实时荧光定量PCR方法检测CLasPrx基因在柑橘不同组织中的表达模式,并在本氏烟Nicotiana benthamiana叶肉细胞瞬时表达CLasPrx分析其编码蛋白的亚细胞定位,利用碘化钾法测定 CLasPrx 蛋白清除 H₂O₂的活性。结果显示,克隆得到的CLasPrx基因序列全长为534 bp,编码177个氨基酸;CLasPrx蛋白包含1个Redoxin保守结构域;多重比对分析发现CLasPrx蛋白活性位点含有保守基序PGAFTPTC;系统发育分析表明CLasPrx蛋白与近缘物种的同源蛋白聚为一类;CLasPrx基因在感染黄龙病柑橘树秋梢中的表达水平显著高于在春梢中的表达水平;CLasPrx定位于本氏烟叶肉细胞的细胞质基质中;过表达CLasPrx蛋白能够显著降低本氏烟叶肉细胞内H₂O₂的含量。表明CLasPrx可能参与柑橘黄龙病菌清除H₂O₂的过程。     

水稻条斑病菌hrpD5基因在致病性中的功能分析

水稻条斑病菌(Xanthomonas oryzae pv.oryzicola,Xoc)成功侵染水稻主要依靠其III型分泌系统(Type III secretion system,T3SS)分泌的效应蛋白。T3SS由hrp-hrc-hpa基因编码,其中主要的hrp和hrc基因突变,病原菌将丧失在寄主水稻上的致病性和非寄主烟草上的过敏性反应(hypersensitive response,HR)。hrpD5基因位于hrp基因簇hrpD操纵单元的第5个基因,在致病性中的功能未知。本研究构建了Xoc的hrpD5缺失突变体RΔhrpD5。植物接种试验显示,RΔhrpD5丧失了对寄主水稻的致病性和在非寄主烟草上激发HR反应的能力;功能互补子虽然能够恢复这2种表型,但是,与野生型菌株相比,其在感病水稻上的毒性显著降低,在非寄主烟草上形成延迟的HR反应;荧光定量PCR结果显示:hrpD5缺失影响其下游hrpD操纵单元基因hrpD6、hrpE和hpaB以及HrpX操纵单元基因hrpF和hrpB1的表达;同时,hrpD5缺失降低了hrpX的mRNA水平,但是不影响hrpX的启动子活性;酵母双杂交结果显示,HrpD5蛋白能够与HrpF蛋白的N端互作。这些结果暗示在Xoc中hrpD5不仅为主要的致病相关基因,而且调控主要的hrp调节基因hrpX的表达。HrpD5新功能的鉴定将为解析T3SS在致病性中的功能提供线索。     

MfMel1基因参与调控桃褐腐病菌对桃果实的致病力

为明确MfMel1基因在桃褐腐病菌Monilinia fructicola中的功能,利用生物信息学软件对该基因的结构、所编码蛋白的保守结构域以及进化关系进行分析;通过遗传转化方法对该基因进行敲除,并进行表型分析。结果显示,敲除转化子△MfMel1菌株的菌丝生长速率、孢子萌发率与野生型菌株Bmpc7均无显著差异,但产孢量显著降低。△MfMel1菌株在0.01%SDS、600μg/mL刚果红、150 g/L甘油、200 g/L蔗糖、6 mmol/L H2O2、4%NaCl和1.2 mmol/L山梨醇培养基上的生长速率、菌丝形态均无显著变化,但对200 g/L葡萄糖的渗透胁迫敏感性增强。敲除转化子△MfMel1菌株的α-半乳糖苷酶活性明显降低,对桃果实的致病力显著下降。表明MfMel1基因在桃褐腐病菌中可能通过参与调控α-半乳糖苷酶酶活,从而参与病原菌的致病。     

UeRbf1调控菰黑粉菌丝状生长和致病力的作用研究

 基于菰黑粉菌的全基因组序列,克隆得到了UeRbf1(GenBank登录号:MW375682),该基因的开放阅读框为1 281 bp,无内含子,编码426 个氨基酸,经预测具有3个C₂H₂锌指结构域,提示其具有转录调控功能。进一步在菰黑粉菌中敲除UeRbf1后进行体外生长及侵染试验,发现:在体外培养时,UeRbf1的缺失没有改变菌株的生长速率及单倍体形态,也未改变两个性亲和UeRbf1缺失突变体的融合及菌丝形成能力,但是融合菌丝的丝状生长能力显著减弱,而且在侵染时UeRbf1缺失突变体丧失了菌丝形成及丝状生长能力,人工接种野茭后也无法使其茎部膨大。以上结果表明,UeRbf1是调控菰黑粉菌丝状生长和侵染能力的一个重要因子。另外, UeRbf1缺失突变后,在体外融合及侵染时相关丝状生长和致病力的调控因子Biz1、Clp1、Hdp1和UeKpp6呈下调表达,其中Biz1的下调表达最为显著,在突变体中几乎不表达,说明UeRbf1对菰黑粉菌丝状生长和致病力的影响,可能是通过调控Biz1来实现的。     

菰黑粉菌MAPK同源基因UeKss1的克隆及表达

菰黑粉菌作为茭白植株体内的内生真菌,其二型态转换与茭白孕茭密切相关,而MAPK途径在真菌二型态转换中具有重要调控作用。本研究在菰黑粉菌中克隆得到了一个MAPK基因UeKss1,通过与酵母菌中的MAPK途径蛋白进行聚类分析表明其属于Kss1的同源蛋白。进一步的系统进化分析表明,UeKss1与黑粉菌属真菌的Kss1同源性最高,达80%以上,且它的丝/苏氨酸双特异性蛋白激酶催化结构域高度保守。不同碳源诱导下菰黑粉菌的生长特征及UeKss1表达分析发现,只有蔗糖培养基能诱导菰黑粉菌菌丝形成,而在PDA、葡萄糖和麦芽糖培养基中,该菌都保持酵母型生长;UeKss1基因的表达量在PDA和葡萄糖培养基中变化不显著,但在麦芽糖培养基中,该基因的表达随着培养时间的延长持续上调表达,而在蔗糖培养基中,UeKss1的表达量虽然也呈现上升趋势,但是远小于麦芽糖的诱导表达量。对UeKss1进行的原核表达纯化,经Western blot验证得到纯度较高的UeKss1蛋白。研究结果可为UeKss1基因的功能研究,阐明其在菰黑粉菌二型态转换中的作用机制提供帮助。     

苹果轮纹病菌LAMP快速检测方法的建立

为建立快捷、灵敏检测苹果轮纹病菌Botryosphaeria dothidea的环介导等温扩增(loop-medi‐ated isothermal amplification,LAMP)检测方法,以其内转录间隔区ITS序列为靶标,设计6条LAMP引物,对其特异性进行检测,优化反应条件并建立苹果轮纹病菌的LAMP检测方法。引物特异性检测结果表明,2株苹果轮纹病菌反应结果呈绿色为阳性,而其它3株对照菌株反应结果呈橙色为阴性,表明了LAMP检测引物的高特异性。优化后的LAMP最佳反应条件:温度65℃、扩增时间60 min、FIP/BIP、F3/B3、LF/LB引物终浓度分别为1.0、0.25、0.5μmol/L。LAMP检测方法对苹果轮纹病菌DNA的检测灵敏度达到了100 ag/μL,是常规PCR检测灵敏度的100倍。田间疑似轮纹病组织检测结果发现LAMP方法对苹果轮纹病菌的检出率高达68%,而传统分离鉴定方法的检出率仅为24%。表明所建立的苹果轮纹病菌LAMP快速检测方法简便快捷、特异性好、灵敏度高,尤其适用于基层植保机构对于苹果轮纹病菌的田间快速检测。     

Development and evaluation of an enzyme-linked immunosorbent assay (ELISA) for the detection of loose smut of barley (Ustilago nuda)

Polyclonal antibodies were raised against mycelium from the logarithmic growth phase of a shake culture of Ustilago nuda, and a double antibody sandwich enzyme-linked immunosorbent assay (ELISA) with biotinylated detection antibodies was developed. The detection limit of the assay was 15 ng total protein ml^–1 for the homologous antigen and 50 ng ml^–1 for a spore extract, respectively. Other species of Ustilago reacted with the antibodies. Cross-reactivity was highest with U. tritici. No signal was obtained with the tested isolates of Tilletia, Rhizoctonia, Pythium and Fusarium. With naturally infected barley seeds, the results of the ELISAs were always in good agreement with those obtained with the routinely used seed embryo test. However, when seeds grown from artificially inoculated florets were used, the ELISA indicated significantly higher infestation levels than the embryo test. Results of assays with halved seeds from the same lot showed that high amounts of mycelium were present in the non-embryo half. This and especially the relatively long duration of the assay suggested that the ELISA (as conducted here) may not be suitable as a routine method for analysing seed infection with U. nuda. With samples from barley seedlings grown from infected seeds the results of the immunoassay again corresponded very well with the infection level determined by staining of the seed embryo, irrespective of the mode of floret inoculation (natural or artificial). Potential fields of application of the ELISA include the early prediction of the efficacy of protection agents, e.g. in screenings for seed treatments, the elucidation of the biology of the fungus and characterisation of resistance mechanisms.     

Fusarium temperatum, a mycotoxin-producing pathogen of maize

In a recent study, a population of Fusarium strains isolated from maize in Belgium was described as a new species, F. temperatum, that is morphologically similar and phylogenetically closely related to F. subglutinans, a species in the American clade of the Gibberella fujikuroi species complex. In fields, the F. temperatum:F. subglutinans ratio was very high, suggesting that F. temperatum outcompetes its sister species F. subglutinans. This raised the question whether this novel species contributes to the final rot symptoms observed on maize plants at harvest, as well as to the potential mycotoxin contamination. Results of the pathogenicity tests by soil and toothpick inoculation demonstrate the ability of F. temperatum to cause seedling malformation and stalk rot under greenhouse conditions. Screening of 15 Fusarium mycotoxins showed the ability of F. temperatum to produce moniliformin, beauvericin, enniatins and fumonisin B₁. The results indicate that F. temperatum can produce mycotoxins and cause maize diseases and, therefore, poses a potential risk to maize production and to the safety of human food and animal feed.     

效应因子PsCRN77基因在本氏烟中的表达降低其对寄生疫霉的抗性

CRN(Crinkler)是一类疫霉菌胞内效应因子,绝大多数CRN的功能和作用机制尚不明确。本研究利用农杆菌介导的瞬时表达技术,发现大豆疫霉效应因子PsC RN77可以在本氏烟中抑制激发子诱导的细胞死亡。接种分析表明在本氏烟中表达该效应因子基因可以促进寄生疫霉的侵染。定量RT-PCR结果显示在本氏烟中表达PsCRN77可以负调控水杨酸信号途径标记基因PRb-1b的表达,正调控茉莉酸信号途径标记基因LOX的表达。进一步通过DAB染色表明,与表达GFP对照相比,表达PsCRN77可以降低疫霉菌侵染后本氏烟中的活性氧积累。以上结果表明PsCRN77是一个毒性效应因子,可以通过影响激素抗性信号途径和活性氧积累干扰植物的防卫反应,促进病原菌的侵染。该研究为认识疫霉菌的致病机制提供了线索。