板栗疫病菌致病力分化的研究

中国板栗(Castanea mollissima Blume)对板栗疫病菌(Cryphonectria parasitica(Murr.)Barr)具有强的抗病力。但近十多年来,我国许多省份均发现有板栗疫病,据调查,广西有19个县市存在栗疫病,有的地方发病还相当严重。为了解释疫病发生的上述情况,并为生产和该病菌的更深入研究提供指导,作者在广西桂林、南宁、柳州、梧州、河池等地收集菌株,对疫病菌致病性的分化进行了研究,现将结果报道如下。 1 材料和方法 1.1 毒力参照菌株 法国已知弱毒株EPF为毒力参照株。 1.2 栗疫病菌的采集、分离和单孢纯化 1.2.1 标本采集:1988年12月～1989年6月,采自广西桂林、南宁、柳州、梧州、河池等地板栗病株。 1.2.2 菌株分离和纯化:将病枝或病树皮,用经灯焰灼烧过的刀片削去表皮,取坏死内皮约5×5mm^2,压于PDA上,28℃,光照培养,产孢后用微块法进行单孢纯化。 1.3     

板栗疫病菌ntl基因的功能研究

板栗疫病菌(Cryphonectria parasitica)是引起板栗疫病的病原真菌。在实验室前期研究中,获得了一个不支持病毒复制且丧失致病力的板栗疫病菌紫外诱变突变株UV57。与野生型菌株EP155相比,UV57中检测不到蛋白100472的表达。为研究蛋白100472的功能,我们构建了编码100472蛋白的ntl基因的缺失突变体△ntl及其互补转化株△ntl-com。与野生型EP155相比,△ntl菌株表型不变,但对休眠板栗树枝的致病性明显降低,而互补转化株△ntl-com的致病力与野生型没有区别。ntl基因的缺失不影响低毒病毒CHV1-EP713的复制累积量,但导致参与G蛋白信号传导途径的cpga1、cpgb1、cpgc1和ste12基因以及参与MAPK途径的cpmk1转录水平明显下调。本研究结果为阐明病原真菌致病机制提供了新的知识。     

板栗疫病菌cpnat基因的功能分析

赖氨酸乙酰化是一种重要的蛋白质翻译后修饰,广泛参与多种生命过程的调节.目前,赖氨酸乙酰化修饰在植物病原真菌——板栗疫病菌中的功能和调节机制尚无报道.本研究克隆了板栗疫病菌的编码乙酰转移酶的cpnat基因,成功构建了cpnat基因的缺失突变体Δcpnat,与野生型菌株相比,板栗疫病菌cpnat基因缺失株生长速率不变,气生...     

广东省水稻纹枯病菌遗传多样性与致病力分化的研究

利用随机引物扩增多态性DNA（PAPD）分子标记技术分析了来自广东省7个县市48个水稻纹枯病菌菌株的遗传多样性,以筛选出的10个随机引物对菌株进行PAPD－PCR扩增,共产生了98个PAPD分子标记,其中89．9％的片段具有多态性。48菌株间的遗传相似性系数（以Nei基因一致度表示）在0．56－0．949之间,用UPGMA和类分析可将它们分为5个RAPD遗传聚类群（A、B、C、D、E）,相同地区来源的菌株基本上聚类在同一组群内。在温室中对供试菌株进行致病性测定,结果表明所有菌株对水稻品种Tetep都有致病性,菌株间致病力差异显著（α＝0．05）,病情指数范围为0．73－18．7,平均感指病指数为5．24。试验分析结果表明水稻纹枯病菌具有丰富的遗传多样性,不同县市的菌株存在很大的遗传分化现象（FST＝0．579）,RAPD遗传聚类群的划分与菌株的地理来源有明显的相关性,但菌株的致病力差异与菌株的来源、遗传聚类组群的划分没有明显的相关性。     

大白菜软腐菌种群组成及优势菌致病型的研究

对黑龙江省成熟大白菜[Brassica pekinensis（Lour．）Rupr．]生产田采集的软腐病菌进行分离、纯化,并根据形态学特征分析了大白菜软腐病菌的种群组成。结果表明,引起黑龙江省秋季大白菜软腐病的主要致病菌是胡萝卜软腐欧文氏菌胡萝卜软腐亚种[Erwinia carotovora（Jones）Bergey et al．subsp、carotovora]（Ecc）;利用20个Ecc菌株的混合菌对来源于不同生态型及不同地区的大白菜品种进行接种,筛选出5个鉴别寄主,以此将20个Ecc菌株划分为5个致病力类型,其中V型为优势致病菌,其分布广且致病力强。     

中国东部栗疫病菌群体的遗传分化

本实验采用RFLP技术,对中国东部栗疫病菌(Cryphonectria parasitica)进行了群体遗传结构的研究。313个参试菌株来自10个省(市)的16个群体(子群体),样本分布在北纬24°N—41°N。各菌株的DNA分别用限制性内切酶Pst Ⅰ和EcoR Ⅰ酶切,先后以10个低拷贝DNA探针和1个DNA指纹图谱探针进行了杂交和检测。结果表明,两个探针(pCB29和pMS29.1)的杂交图谱呈单态性;探针pCB19的杂交图谱显示,菌株DNA以PstⅠ酶切的为单态性,以EcoR Ⅰ酶切的则呈多态性;其他7个低拷贝探针的杂交图谱都呈多态性(Pst Ⅰ酶切)、指纹图谱探针的检测结果显示,辽宁凤城群体的菌株与中国东部其他群体的菌株相比,具有更多的限制性杂交片段,菌株间的遗传变异性也更大。     

沙门菌噬菌体抗性菌的筛选鉴定及致病力研究

【目的】筛选鉴定沙门菌噬菌体侵染裂解过程中的抗性菌株,研究抗性菌株的生物学特性及致病力的差异,为解决噬菌体治疗应用中的抗性菌问题提供理论依据。【方法】本研究通过次级感染法和双层平板法筛选沙门菌噬菌体抗性菌,通过生物学特性和毒力基因检测比较宿主菌ATCC 13076及其噬菌体抗性菌株R3之间的差异,并通过小鼠攻毒实验和细胞粘附实验比较致病力强弱。【结果】噬菌体抗性菌株R3的生长速度较宿主菌略慢;生化及毒力基因检测均表明抗性菌株与宿主菌无差异;与宿主菌相比,抗性菌R3的LD50增加了74.8%(P>0.05);对MODE-K细胞粘附能力稍弱,但是差异不显著。【结论】该研究表明,与噬菌体宿主菌相比,噬菌体抗性菌株的生物学特性和毒力基因并没有改变,对小鼠致病力减弱,但是对MODE-K细胞粘附能力差异不显著。     

亚欧美栗疫病菌群体的遗传多样性

从 12 0个随机引物中筛选出条带清晰、主带明显、重复性好的 9个引物 ,对来自不同地域和寄主的 7个群体的 14 2个栗疫病菌菌株进行 RAPD分析。 9个引物共扩增出条带 12 4条 ,其中多态性条带 111条 ,多态性比率为 89.5 2 %。利用 Popgen3.2软件对供试群体进行遗传多样性分析和 UPGMA聚类。结果表明 ,中国地区 4个群体间的遗传相似性较大 ,与美国、意大利和日本群体间的相似性较小 ;美国和意大利群体间的遗传相似性较大 ,且它们与日本群体间的相似性大于与中国群体间的相似性。病原菌群体的遗传变异率为 0 .2 35 1,其中在地区水平上 ,82 .34%由群体内的变异引起 ,17.6 6 %由群体间的差异引起 ,群体间的基因流动值为 2 .3311;而在寄主水平上 ,则 79.4 2 %由群体内的变异引起 ,2 0 .5 8%由群体间的差异引起 ,群体间的基因流动值为 1.92 97     

番茄青枯雷尔氏菌强致病力菌株变异的研究

为研究番茄青枯雷尔氏菌强致病力菌株的变异,探索了继代培养、在NB培养基上不同时间培养、不同pH处理7d和15d、不同温度处理1h后对强致病力菌株变异的影响。结果表明:随着继代培养的培养代数增加,平均弱化指数成增大趋势,在第10代出现了无致病力菌株;在NB培养基上培养15d时,强致病力菌株已完全转化为不确定菌株和无致病力菌株,在培养30d时,强致病力菌株几乎完全转化为无致病菌株;pH7.0时,处理7d和15d后,强致病力菌株比例均为最大,分别为93.33%和92.22%,pH5.8时,强致病力菌株比例最低,分别为46.67%和31.11%;用不同温度处理强致病力菌株发现,温度50℃时,菌株死亡,温度40℃时,活菌数显著低于其他(4~30℃)处理,强致病力菌株比例为4~40℃所有处理中最低。     

三株金龟子绿僵菌对红火蚁的致病力测定

本研究在实验室条件下测定了金龟子绿僵菌M09、CQMa117和CQMa128三个菌株对红火蚁的侵染能力。结果表明在1.0×10~8孢子/mL浓度下M09、CQMa128和CQMa117菌株处理后10 d红火蚁的工蚁累计死亡率分别为73.3%、14.6%和55.5%;M09菌株对工蚁的LC50为3.50×106孢子/mL,8.0×107孢子/mL浓度处理LT50为4.35 d。对M09菌株侵染红火蚁幼虫和蛹的能力测定结果显示,1.0×10~8孢子/mL浓度处理后10 d该菌株对幼虫和蛹的累计侵染率为98.9%和100%,对幼虫和蛹LC50分别为6.34×10~4孢子/mL、1.01×10~4孢子/mL。以上研究证实金龟子绿僵菌M09菌株对红火蚁具较强的致病力,致死速度较快,可作为该蚁生物防治的候选菌株。     

土壤中辣椒疫霉分离方法的研究与量化测定

从杭州、西安、广州及武汉等辣椒病田分别采集土样 ,室内晾干研碎后 ,用选择性培养基 ,采用土壤稀释平板法和组织诱饵法分离辣椒疫霉 (PhytophthoracapsiciLeonian) ,并对土壤中辣椒疫霉的密度进行量化处理。结果表明 ,利用选择性燕麦培养基 ,采用土壤稀释平板法可分离获得大量的辣椒疫霉菌株 ,而且辣椒连作田的辣椒疫霉菌密度高于轮作田。组织诱饵法试验结果表明 ,辣椒叶片诱集效果最好 ,其次是辣椒果实。     

Biological Control of Oomycete Soilborne Diseases Caused by Phytophthora capsici,Phytophthora infestans,and Phytophthora nicotianae in Solanaceous Crops

Oomycete pathogens that belong to the genus Phytophthora cause devastating diseases in solanaceous crops such as pepper, potato, and tobacco, resulting in crop production losses worldwide. Although the application of fungicides efficiently controls these diseases, it has been shown to trigger negative side effects such as environmental pollution, phytotoxicity, and fungicide resistance in plant pathogens. Therefore, biological control of Phytophthora-induced diseases was proposed as an environmentally sound alternative to conventional chemical control. In this review, progress on biological control of the soilborne oomycete plant pathogens, Phytophthora capsici, Phytophthora infestans, and Phytophthora nicotianae, infecting pepper, potato, and tobacco is described. Bacterial (e.g., Acinetobacter, Bacillus, Chryseobacterium, Paenibacillus, Pseudomonas, and Streptomyces) and fungal (e.g., Trichoderma and arbuscular mycorrhizal fungi) agents, and yeasts (e.g., Aureobasidium, Curvibasidium, and Metschnikowia) have been reported as successful biocontrol agents of Phytophthora pathogens. These microorganisms antagonize Phytophthora spp. via antimicrobial compounds with inhibitory activities against mycelial growth, sporulation, and zoospore germination. They also trigger plant immunity-inducing systemic resistance via several pathways, resulting in enhanced defense responses in their hosts. Along with plant protection, some of the microorganisms promote plant growth, thereby enhancing their beneficial relations with host plants. Although the beneficial effects of the biocontrol microorganisms are acceptable, single applications of antagonistic microorganisms tend to lack consistent efficacy compared with chemical analogues. Therefore, strategies to improve the biocontrol performance of these prominent antagonists are also discussed in this review.     

辣椒疫霉菌果胶裂解酶PL101基因的克隆及生物信息学分析

【背景】辣椒疫病是一种世界性土传病害,严重影响世界各国辣椒生产,并带来巨大经济损失。果胶裂解酶(pectate lyase,PL)作为一类重要的细胞壁降解酶类是该病的重要致病因子。【目的】对果胶裂解酶基因进行克隆,并对其生物信息学特性进行相关分析,进一步阐明该酶的作用机制。【方法】根据辣椒疫霉菌全基因组序列,以高致病菌株SD33为模板扩增PL101基因的全长cDNA序列,并对其理化性质、跨膜区、亲疏水性、结构域等生物学特性进行分析。【结果】除获得PL101相关生物学特性信息外,还对PL101进行三维结构建模,获得可信度较高的蛋白结构,并确定PL101可能的催化位点为Asp183、Arg212、Arg272三个氨基酸。【结论】对PL101基因的克隆及相关生物学特性的分析为进一步阐明PL功能特性提供参考。     

优质抗疫病甜椒种质资源的选育

本文利用国外引进的2个抗疫病的商业品种,通过系谱法选择,获得了6个园艺性状普遍优于茄门、对疫病达到抗病和高抗水平的株系,其中有4个株系兼中抗CMV和TMV,另有1个株系兼抗CMV.其中20079-0-3-1-27和20080-0-1-3-29综合表现尤其突出.开展优质抗疫病甜椒种质资源的选育,将为国内抗疫病的甜椒育种起到积极的作用.     

Molecular Distinctions Between Phytophthora capsici and Ph. tropicalis Based on ITS Sequences of Ribosomal DNA

Among four species of Phytophthora tested, only Ph. capsici and Ph. tropicalis showed the same length for DNA sequence for both internal transcribed spacer (ITS)1 and ITS2 of ribosomal DNA. Phytophthora palmivora and P. nicotianae have lengths different from each other, and from the other two species. Although A1 and A2 types of Ph. capsici differ from each other by only one nucleotide, there are 10 different nucleotides between A1 and A2 types of Ph. tropicalis. Phylogenetic analysis of combined ITS sequences identified four clades each consisting A1 and A2 mating types of same species. The neighbor‐joining and maximum parsimony trees show that Ph. tropicalis (A2) is clustered with the clade of two isolates of Ph. capsici before joining the clade of A1 and two other isolates of Ph. tropicalis from GenBank. Our results support the separation of Ph. tropicalis and demonstrate the need to sequence more than a single isolate of a species in the study of molecular phylogeny of Phytophthora. The phylogenetic trees also suggest that Ph. tropicalis (A2) may represent a transitional isolate in the process of species evolution.     

拮抗辣椒疫病菌的红树内生细菌筛选及RS261菌株鉴定

红树内生细菌分离及拮抗辣椒疫霉(Phytophthora capsici)筛选结果表明：各红树体内均有大量的内生细菌, 不同红树种类及部位内生细菌的数量均不同, 被测定的红树内生细菌中约有27.97%的可培养菌株对辣椒疫霉具有拮抗作用; 其中18株拮抗作用较强的细菌在辣椒果上对辣椒疫病菌均有一定的抑制效果, 以来自红海榄叶片内的RS261菌株效果最好; 经形态、生理生化特征和分子生物学等测定分析, 将RS261菌株初步鉴定为解淀粉芽孢杆菌(Bacillus amyloliquefaciens)。     

Inhibitory Effect of Paenibacillus polymyxa GBR-462 on Phytophthora capsici Causing Phytophthora Blight in Chili Pepper

In the present work 25 strains of Paenibacillus polymyxa isolated from rotted ginseng roots were screened for their antimicrobial activity against Phytophthora capsici in vitro . Based on antimicrobial activity, 15 strains categorized as strongly antimicrobial, among them GBR-462 was found as the most active, and five strains each as weekly antimicrobial and no antimicrobial. Antimicrobial activity was influenced by the initial inoculum density, as strains of P. polymyxa with a strong antimicrobial activity (including P. polymyxa GBR-462) showed the antimicrobial activity against P. capsici and could form biofilm only when they were applied at the higher initial inoculums, 10⁸ cfu/ml. No inhibitory effect was noted on the mycelial growth and zoospore germination of the pathogen when applied at the lower inoculum density of 10⁶ cfu/ml of P. polymyxa GBR-462. However, sporangium formation and zoospore release was significantly inhibited at the lower inoculum density. Also light and electron microscopy revealed the structures of sporangia aberrant with no or few healthy nuclei, indicating sporangium and zoospore formation inhibited at the lower inoculum density. Application of P. polymyxa GBR-462 into potted soil suppressed disease progression as well as disease severity; disease severity was reduced by 30% as compared to untreated pots, suggesting P. polymyxa GBR-462 could be a potential biocontrol agent against Phytopthora capsici .     

辣椒疫霉菌侵染模型和侵染条件定量研究

在生长箱内控制条件下分析测定了土壤温度、水分含量对辣椒疫病死苗率的影响.结果表明:土壤温度和水分状况是决定辣椒疫病菌侵染的重要因子,病菌侵染的最适土壤温度为22 ℃～28 ℃,最适土壤含水量为40%,土壤过于干燥和过饱和都不利于病菌侵染发病;辣椒疫病死苗率与土壤温度、水分含量及其互作可用数学模式描述.田间调查发现,辣椒疫病田间流行趋势可用Gompertz模型描述,发病率与初始发病率、土壤温度、水分含量以及空气温度密切相关.建立了田间辣椒疫病发病率预测模型.     

辣椒感染疫病后生化指标的响应研究

采用生理生化分析方法研究了辣椒感染疫病后叶片中几个生化指标的变化。结果表明,染病前后感病品种叶片中可溶性总糖含量持续高于抗病品种;抗病类型品种和感病类型品种的可溶性蛋白含量变化规律均表现为先升高后下降,但接种前其叶片中可溶性蛋白含量两者间无明显差异;抗病类型和感病类型辣椒接种后保护酶活性均升高,而且感病类型的POD和ASP酶活性在接种后120h显著高于抗病类型;高抗类型叶片中PPO活性增加幅度显著大于感病类型,但抗病类型品种PPO活性上升趋势比较平缓。接种后,感病品种PAL活性上升幅度小于高抗品种,接种后96h PAL活性开始逐渐下降。可溶性总糖含量和苯丙氨酸解氨酶可以作为辣椒苗期抗疫病鉴定的生化指标。     

Proteomic profile of the plant‐pathogenic oomycete Phytophthora capsici in response to the fungicide pyrimorph

Pyrimorph is a novel fungicide from the carboxylic acid amide (CAA) family used to control plant‐pathogenic oomycetes such as Phytophthora capsici. The proteomic response of P. capsici to pyrimorph was investigated using the iTRAQ technology to determine the target site of the fungicide and potential biomarker candidates of drug efficacy. A total of 1336 unique proteins were identified from the mycelium of wild‐type P. capsici isolate (Hd3) and two pyrimorph‐resistant mutants (R3‐1 and R3‐2) grown in the presence or absence of pyrimorph. Comparative analysis revealed that the three P. capsici isolates Hd3, R3‐1, and R3‐2 produced 163, 77, and 13 unique proteins, respectively, which exhibited altered levels of abundance in response to the pyrimorph treatment. Further investigations, using Cluster of Orthologous Groups of Proteins (COG) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis identified 35 proteins related to the mode of action of pyrimorph against P. capsici and 62 proteins involved in the stress response of P. capsici to pyrimorph. Many of the proteins with altered expression were associated with glucose and energy metabolism. Biochemical analysis using d ‐[U‐¹⁴C]glucose verified the proteomics data, suggesting that the major mode of action of pyrimorph in P. capsici is the inhibition of cell wall biosynthesis. These results also illustrate that proteomics approaches are useful tools for determining the pathways targeted by novel fungicides as well as for evaluating the tolerance of plant pathogens to environmental challenges, such as the presence of fungicides.