外源合成dsRNA介导大豆疫霉PsCdc14基因沉默后对孢子囊发育的影响

真核生物中,蛋白磷酸酶Cdc14在细胞有丝分裂过程中发挥着重要的调控作用.已有研究表明,在导致马铃薯晚疫病的致病疫霉病原菌中,Cdc14对游动孢子囊的形成过程起关键性的调控作用.但其在导致大豆根腐病的大豆疫霉病原菌中的作用机制还不清楚.实时定量PCR分析大豆疫霉PsCdc14在不同的生活史和侵染阶段的转录水平发现,PsCdc14在游动孢子囊形成、游动孢子和休止孢3个阶段具有较高的转录水平,但是在菌丝和侵染阶段转录水平很低.由此推测,PsCdc14在疫霉无性繁殖阶段发挥着重要的调控作用,进而影响病害的传播及蔓延.双链RNA（dsRNA）介导的转录后目的基因沉默是真核生物十分保守的一种调控机制.将体外合成dsRNA和聚乙二醇（PEG）介导的大豆疫霉原生质体转化技术相结合,建立了大豆疫霉dsRNA介导的瞬时基因沉默体系,并利用此体系对大豆疫霉中PsCdc14基因进行了功能分析.结果表明,体外合成的PsCdc14dsRNA转入大豆疫霉原生质体后,能够导致内源的基因转录水平显著下降,沉默转化子的游动孢子囊产量显著降低.本研究表明,利用dsRNA介导的瞬时基因沉默能够更加方便、快捷地分析大豆疫霉的基因功能,加深人们对大豆疫霉生长发育和致病机制的理解.     

虫霉目新种和新记录

本文报道了虫霉目的一个新种和3个新记录种。新种叶蝉虫疫霉(Erynia cicadellisLi et Fan sp.nov.)是我国南方稻叶蝉虫霉流行病的病原之一,与近缘种飞虱虫疫霉[E．Deiphaeis (Heri) Humber et Ben-Ze’ev] 的主要区别在于后者孢子较大,且不具有假根。蚜虫疫霉[E. aphidis (Hoffm.) Humber et Ben-Ze'ev]系世界不常见种,国内首次在陕西岚皋及福州的桃蚜上记录;飞虱虫疫霉和佩氏虫疫霉[E．Petchii Ben-Ze'ev et Kenneth)皆系南方褐稻虱虫霉流行病的病原。     

福建、浙江、江苏、上海疫霉种的研究

1985—1986年,从福建漳州,浙江杭州,上海,江苏南京,苏州,扬州,常州等地采集100多种植物进行分离,在雪松(Cedrus deodara),倒挂仙人鞭(Cereus flagelliformis),冬青卫茅(Euonymus japonieus),构树(Broussonetia papyrifera),蓖麻(Ricinus communis),柑桔(Citrus reticulata),带叶兜兰(Paphiopedilum hirsutissimum),芋(Colocasia esculenta),鳄梨(Persea americana),山茶(Camellia sp.),珊瑚樱(Solanum pseudo-capsicum),百合(Lilium brownii var.viridulum),蔓常春花(Vinca major),西瓜(Citrullus lanatus)等14种寄主植物上分离到51个疫霉菌株,鉴定为9个种:烟草疫霉(=寄生疫霉)Phytophthora nicotianae B.de Haan(=P.parasitica Dast.),掘氏疫霉P.drechsleri Tucker,樟疫霉 P.ciunamomi Rands,棕榈疫霉P.palmivora(Butl.)Butl.,柑桔褐腐疫霉P.citrophthora(R.et E.Smith) Leon.,苎麻疫霉P.bochmeriae Saw.,蜜色疫霉P.meadii McRae,簇囊疫霉P.botryosa Chee,隐地疫霉P.cryptogea Pethyb.et Laff.。其中,烟草疫霉,掘氏疫霉,樟疫霉出现频率较高,分布较广,初步认为是上述地区植物疫病的主要病原菌。簇囊疫霉、蜜色疫霉是国内新记录。     

恶疫霉致病力和对甲霜灵敏感性的遗传与变异

以分离自黑龙江腐烂苹果的恶疫霉Phytophthora cactorum Schroeter野生型菌株Ap14为亲本,采用菌丝块创伤接种法测定了恶疫霉对苹果的致病力在无性繁殖和有性生殖后代的遗传,结果是连续2代单游动孢子后代在苹果上所致病斑半径分别为22.4-24.1mm和21.8-23.4mm,与亲本所致病斑半径22.5mm无显著差异;而其20个自交后个体所致病斑半径为21.4-25.8mm,与亲本有显著差异。其中2株致病力显著强于亲本,其余与亲本相似,表明恶疫霉对苹果的致病力在无性后代可稳定遗传,而在自交有性后代发生分离,同时在含甲霜灵0.05μg/ml的LBA平板上测定了恶疫霉菌丝生长对甲霜灵的敏感性在无性和有性后代的遗传。结果是恶疫霉对甲霜灵的敏感性在无性单孢后代无显著变异,而在50个自交有性后代中,上述浓度对其菌丝生长的抑制率分布范围为67.3-97.1%,与亲本78.3%有极显著差异。其中3株高于亲本,3株低于亲本,44株与亲本相似,表明恶疫霉对甲霜灵的敏感性同样在无性后代稳定遗传而在有性后代发生变异,上述结果提示,供试恶恶疫霉菌株中上述性状可能由细胞核杂合基因控制。     

核糖体基因ITS作为苎麻疫霉、恶疫霉分类辅助性状的研究*

以2个雄器大多围生、少数侧生的苎麻疫霉菌株与1个雄器侧生、偶有围生的恶疫霉菌株为材料,采用真菌核糖体基因转录间隔区（ITS）通用引物,PCR扩增3个供试菌株核糖体基因的ITS1和ITS2,并对PCR产物进行了克隆和序列分析。结果是苎麻疫霉的ITS1和ITS2分别由206和453个碱基组成, 而恶疫霉则分别由218和415个碱基组成。2个供试苎麻疫霉菌株的ITS1和ITS2的碱基序列同源性均分别为100%。苎麻疫霉和恶疫霉ITS1同源性为74.9%,其中中间区域40bp-164bp之间在两种间变异丰富,同源性只有59.4%,而1bp-39bp和165bp-239bp两区域的同源性分别为92.3%和92.1%; ITS2在两种疫霉菌间的同源性为71.0%。结果表明苎麻疫霉和恶疫霉ITS的碱基序列有明显差异。上述结果提示,ITS区域碱基序列可区分苎麻疫霉和恶疫霉。     

核糖体基因ITS作为苎麻疫霉、恶疫霉分类辅助性状的研究

以2个雄器大多围生、少数侧生的苎麻疫霉菌株与1个雄器侧生、偶有围生的恶疫霉菌株为材料,采用真菌核糖体基因转录间隔区（ITS）通用引物,PCR扩增3个供试菌株核糖体基因的ITS1和ITS2,并对PCR产物进行了克隆和序列分析。结果是苎麻疫霉的ITS1和ITS2分别由206和453个碱基组成,而恶疫霉则分别由218和415个碱基组成。2个供试苎麻疫霉菌株的ITS1和ITS2的碱基序列同源性均分别为100%。苎麻疫霉和恶疫霉ITS1同源性为74.9%,其中中间区域40bp-164bp之间在两种间变异丰富,同源性只有59.4%,而1bp-39bp和165bp-239bp两区域的同源性分别为92.3%和92.1%;ITS2在两种疫霉菌间的同源性为71.0%。结果表明苎麻疫霉和恶疫霉ITS的碱基序列有明显差异。上述结果提示,ITS区域碱基序列可区分苎麻疫霉和恶疫霉。     

辣椒疫霉RXLR型效应子PcAvh2的序列多态性、基因转录特征及功能

【目的】分析辣椒疫霉中RXLR型效应子PcAvh2的序列多态性,研究该效应子在辣椒疫霉生长发育和侵染阶段的转录特征及其生物学功能。【方法】本研究通过高保真扩增,分析2个烟草疫霉、1个恶疫霉和31个辣椒疫霉菌株的PcAvh2序列;提取辣椒疫霉菌丝、游动孢子囊、游动孢子、萌发休止孢和7个侵染时间点(1.5、3、6、12、24、36、72 h)的本氏烟根部总RNA,利用RT-qPCR分析PcAvh2的转录表达水平;利用PVX瞬时表达系统,分析PcAvh2是否抑制6种效应子(BAX、INF1、PsojNIP、PsCRN63、PsAvh241、R3a/Avr3a)激发的植物免疫反应;利用CaCl_2-PEG介导的原生质体稳定转化技术,沉默PcAvh2基因,分析辣椒疫霉致病力的变化。【结果】PcAvh2为典型的RXLR效应子,在辣椒疫霉群体中该效应子具有10个等位基因,而且烟草疫霉和恶疫霉中也存在该效应子。该基因在辣椒疫霉的侵染阶段上调表达,它能够抑制6种效应子激发的植物免疫反应,进一步研究发现基因沉默导致辣椒疫霉的致病力显著下降。【结论】RXLR型效应子PcAvh2是辣椒疫霉中一个重要的侵染致病因子。     

中国橡胶树疫霉种的研究*

疫病是我国植胶区的主要病害。近年来,作者从云南西双版纳和广东海南岛的橡胶树和胶园土共分离出57株疫霉菌种。通过分类研究,共鉴定出4个种:恶疫霉 Phytophthoracactorum(Leb.＆ Cohn)Schroeter,辣椒疫霉 P.capsici Leoman,柑桔褐腐疫霉 P.citrophthora(Sm.＆ Sm.)Leonian,和棕榈疫霉 P.palmivora(Butl.)Butler。其中辣椒疫霉是首次在橡胶树上发现。我国橡胶树疫霉的种群结构与东南亚和南亚的有所不同,除棕榈疫霉外,其余3种在东南亚和南亚均未发现。而东南亚常见种:簇囊疫霉(P.botryosa)、橡胶疫霉(P.heveae)和蜜色疫霉(P.meadii),在我国却迄今尚未发现或有待证实。以前报道分离自胶园土壤中的芋疫霉(P.colocasiae),可能系柑桔褐腐疫霉之误。绝大多数分离物经配对培养均可产生性器官:辣椒疫霉的A~1交配型和A~2交配型大致相等;柑桔褐腐疫霉和棕榈疫霉的A~2交配型则明显多于A~1交配型。     

中国橡胶树疫霉种的研究

疫病是我国植胶区的主要病害。近年来,作者从云南西双版纳和广东海南岛的橡胶树和胶园土共分离出57株疫霉菌种。通过分类研究,共鉴定出4个种:恶疫霉 Phytophthoracactorum(Leb.＆ Cohn)Schroeter,辣椒疫霉 P.capsici Leoman,柑桔褐腐疫霉 P.citrophthora(Sm.＆ Sm.)Leonian,和棕榈疫霉 P.palmivora(Butl.)Butler。其中辣椒疫霉是首次在橡胶树上发现。我国橡胶树疫霉的种群结构与东南亚和南亚的有所不同,除棕榈疫霉外,其余3种在东南亚和南亚均未发现。而东南亚常见种:簇囊疫霉(P.botryosa)、橡胶疫霉(P.heveae)和蜜色疫霉(P.meadii),在我国却迄今尚未发现或有待证实。以前报道分离自胶园土壤中的芋疫霉(P.colocasiae),可能系柑桔褐腐疫霉之误。绝大多数分离物经配对培养均可产生性器官:辣椒疫霉的A~1交配型和A~2交配型大致相等;柑桔褐腐疫霉和棕榈疫霉的A~2交配型则明显多于A~1交配型。     

大豆疫霉根腐病抗源筛选

由大豆疫霉菌引起的大豆疫霉根腐病是大豆生产的重要病害,该病已在我国大豆主要产区发生,并在局部地区造成较大产量损失。利用抗病品种是防治大豆疫霉根腐病最有效的方法。本研究目的是筛选大豆疫霉根腐病抗源,为病害防治和抗病品种的选育提供参考。用下胚轴创伤接种方法对120个栽培大豆品种（系）进行接种,鉴定其对10个具有不同毒力大豆疫霉菌菌株的抗性。有110个品种（系）分别抗1～10个大豆疫霉菌菌株,其中以河南大豆品种（系）对疫霉菌的抗性最丰富,安徽、湖北和山西大豆品种（系）也具有抗性多样性。120个大豆品种（系）对10个大豆疫霉菌菌株共产生57个反应型,有4个抗性反应型分别与单个抗病基因的反应型一致,有7个抗性反应型与2个已知基因组合的反应型相同,其他抗性反应型为新的类型。一些大豆品种（系）中可能存在有效的抗大豆疫霉根腐病新基因。     

Rapid Detection of Phytophthora nicotianae in Infected Tobacco Tissues and Soil Samples Based on Its Ypt1 Gene

This paper describes the development of a polymerase chain reaction (PCR) assay for the detection of Phytophthora nicotianae , the causal agent of Phytophthora blight of tobacco and other plants. The PCR primers were designed based on a Ras-related protein ( Ypt 1) gene, and 115 isolates representing 26 species of Phytophthora and 29 fungal species of plant pathogens were used to test the specificity of the primers. PCR amplification with species-specific (Pn) primers resulted in a product of 389 bp only from isolates of P. nicotianae . The detection sensitivity with Pn primers was 1 ng of genomic DNA. Using Ypt 1F/ Ypt 1R as first-round amplification primers, followed by a second round using the primer pair Pn1/Pn2, a nested PCR procedure was developed, which increased the detection sensitivity 100-fold to 10 pg. PCR with the Pn primers could also be used to detect P. nicotianae from naturally infected tobacco tissues and soil. The PCR-based methods developed here could simplify both plant disease diagnosis and pathogen monitoring as well as guide plant disease management.     

马铃薯立枯丝核菌拮抗菌QHZ11的实时荧光定量PCR快速检测与应用

【背景】植物根际促生菌(plant growth-promoting rhizobacteria,PGPR)在根际的定殖是其发挥作用的基础,直观有效的跟踪技术和定量方法是研究PGPR在根际原位分布规律的重要工具。【目的】建立一种马铃薯黑痣病病原菌——立枯丝核菌拮抗菌QHZ11的实时荧光定量PCR快速检测体系,并检测拮抗菌QHZ11在马铃薯根际的动态变化。【方法】根据GenBank中登录的类芽孢杆菌及近源菌株gyrB基因序列差异筛选特异性引物,优化反应条件;通过盆栽试验对马铃薯根际拮抗菌进行快速检测。盆栽试验设3个处理,T1：对照(无菌水,CK);T2：QHZ11菌悬液灌土(QHZ11);T3：将功能菌在有机肥中进行二次固体发酵制成生物有机肥(BOF11)。【结果】筛选出拮抗菌QHZ11的专用引物为gyrB-F/gyrB-R;建立的拮抗菌QHZ11实时荧光定量PCR检测方法特异性好、灵敏度高且重复性较好,线性相关系数为0.999 8,检测组内变异系数均在1%以内,扩增效率为0.9,可检测出1×103?1×1010 copies/g-soil的拮抗菌,具有检出限低和扩增效率高的特点。盆栽试验结果发现,T3处理马铃薯根际拮抗菌QHZ11的数量从接种的第10天即高出T2处理一个数量级,并于马铃薯盛花期(接种后第60天)达到峰值,说明二次固体发酵增加了拮抗菌在根际土壤中的存活和繁殖。【结论】建立的实时荧光定量PCR快速检测体系灵敏、高效,可为研究拮抗菌在马铃薯根际原位分布以及与病原菌的互作方面提供便捷有效的方法。     

Evaluation of fatty acid methyl ester profile and amplified fragment length polymorphism analyses to distinguish five species of Phytophthora associated with ornamental plants

Fatty acid methyl ester (FAME) profiles and amplified fragment length polymorphisms (AFLPs) were evaluated as tools for identifying species of Phytophthora. Five isolates of each of Phytophthora cactorum, Phytophthora citrophthora, Phytophthora cinnamomi, Phytophthora nicotianae and Phytophthora cryptogea were subjected to both analyses to examine variation among and within species. In FAME analysis, isolates of P. cactorum, P. cinnamomi and P.nicotianae were clustered by species, but isolates of P. citrophthora and P.cryptogea were divided into multiple clusters based on greater variations within these two species. The AFLP analysis differentiated all five species of Phytophthora. The five isolates of each species were grouped in a separate terminal cluster, but diversity within a species cluster varied considerably with variation greater in P. cryptogea and P. citrophthora. Comparing the dendrograms based on FAME and AFLP analyses, the overall patterns of both were similar. The P. cactorum cluster was distinct from clusters of the other four species, which formed one large cluster. The higher values of percentages of polymorphic loci and gene diversity in AFLP analysis substantiated diversity observed among isolates of P. citrophthora and P. cryptogea in FAME and AFLP dendrograms. Both FAME and AFLP appear to be useful tools for identifying species of Phytophthora, but only AFLP analysis has potential to study genetic and phylogenetic relationships within and among species in this genus.     

Susceptibility of Thirty Cherry Genotypes on Phytophthora cactorum, P. citrophthora, P. citricola and P. parasitica

The diseases Phytophthor a crown and root rot consist of the most important problems in cherry cultivation. In this study, the susceptibility of 30 cherry genotypes to Phytophthora cactorum , P. citrophthora, P. parasitica and P. citricola was evaluated by using excised twig assay, excised shoot method and stem inoculation method. The results showed that all cherry genotypes tested were susceptible to all Phytophthora isolates used. Phytophthora parasitica and P. citrophthora were the most aggressive species. Host specificity of the Phytophthora isolates used in this study was not found although these isolates were from different plant species. In conclusion, because none of the cherry genotype showed a level of resistance to these pathogens, caution should be taken when these genotypes are used in locations, where these diseases are endemic.     

Single-strand-conformation polymorphism of ribosomal DNA for rapid species differentiation in genus Phytophthora

Single-strand-conformation polymorphism (SSCP) of ribosomal DNA of 29 species (282 isolates) of Phytophthora was characterized in this study. Phytophthora boehmeriae, Phytophthora botryosa, Phytophthora cactorum, Phytophthora cambivora, Phytophthora capsici, Phytophthora cinnamomi, Phytophthora colocasiae, Phytophthora fragariae, Phytophthora heveae, Phytophthora hibernalis, Phytophthora ilicis, Phytophthora infestans, Phytophthora katsurae, Phytophthora lateralis, Phytophthora meadii, Phytophthora medicaginis, Phytophthora megakarya, Phytophthora nicotianae, Phytophthora palmivora, Phytophthora phaseoli, Phytophthora pseudotsugae, Phytophthora sojae, Phytophthora syringae, and Phytophthora tropicalis each showed a unique SSCP pattern. Phytophthora citricola, Phytophthora citrophthora, Phytophthora cryptogea, Phytophthora drechsleri, and Phytophthora megasperma each had more than one distinct pattern. A single-stranded DNA ladder also was developed, which facilitates comparison of SSCP patterns within and between gels. With a single DNA fingerprint, 277 isolates of Phytophthora recovered from irrigation water and plant tissues in Virginia were all correctly identified into eight species at substantially reduced time, labor, and cost. The SSCP analysis presented in this work will aid in studies on taxonomy, genetics, and ecology of the genus Phytophthora.     

Specific and Sensitive Detection of Phytophthora nicotianae by Nested PCR and Loop‐mediated Isothermal Amplification Assays

Phytophthora nicotianae is an important soilborne plant pathogen. It causes black shank in tobacco and other commercially important crop diseases. Early and accurate detection of P. nicotianae is essential for controlling these diseases. In this study, primers based on the Ras‐related protein gene (Ypt1) of P. nicotianae were tested for their specific detection of the pathogen using nested PCR and LAMP assays. For specificity testing, DNA extracts from 47 P. nicotianae isolates, 45 isolates of 16 different oomycetes and 25 isolates of other fungal species were used; no cross‐reaction with other pathogens was observed. The sensitivity assay showed that the nested PCR and LAMP assays had detection limits of 100 fg and 10 fg genomic DNA per 25‐μl reaction, respectively. Furthermore, the nested PCR and LAMP assays were used for the detection of DNA from naturally P. nicotianae‐infected tobacco tissues and soil. Our results suggest that the LAMP assay has the greatest potential for the specific detection of P. nicotianae in regions that are at risk of contracting tobacco black shank disease and that the Ypt1 gene is a novel and effective target of P. nicotianae LAMP visual detection.     

Relative Resistance of Four Peach Rootstocks to Phytophthora cactorum and Phytophthora megasperma

Shoot samples of four peach rootstocks that are important to the peach industry in Greece: KID I, GF305, GF677, and PR204, were inoculated with Phytophthora cactorum and Phytophthora megasperma in the field and in the glasshouse and were then evaluated with regard to susceptibility. The pathogenicity of P. cactorum and P. megasperma to peach rootstocks was confirmed with an excised twig assay. The peach rootstocks showed differential susceptibility to P. cactorum . GF305 was the least susceptible and KID I was the most susceptible, which suggests that the latter rootstock is unsuitable for orchards in which the conditions are favourable for Phytophthora diseases. GF677 and PR204 were moderately susceptible. The plants that were inoculated with P. megasperma in the field and in the glasshouse showed no sign of infection whereas twigs inoculated in vitro with P. megasperma developed necrosis. GF305 was the most resistant, KID I was the most susceptible and PR204 and GF677 were moderately resistant. The present results demonstrate that none of the four peach rootstocks used in this study was completely resistant to P. cactorum , particularly when rootstocks were flooded periodically to enhance disease development. Therefore an integrated approach including host and resistance cultural practices is recommended to manage diseases caused by P. cactorum in peach orchards in Greece.     

Aminopeptidase profiles of four species of phytophthora

Phytophthora palmivora, P. cinnamomi, P. drechsleri, and P. cactorum were readily separated on the basis of the aminopeptidase substrate specificities of their mycelial suspensions. Variability among isolates of the same species was not significant with most substrates and isolates tested, regardless of source. Both qualitative and quantitative differences in enzyme activity were useful for species identification. Differentiation of these four species was possible with comparative reactions of l-alanyl-, l-arginyl-, l-benzoylarginyl, l-gamma-glutamyl-, l-glycyl-, l-hydroxyprolyl-, l-leucyl-, l-lysyl-, 4-methoxyleucyl-, l-prolyl-, and 4-methoxyalanyl-beta-naphthylamides. Variation in peptidase activity was usually detectable after 4 h of incubation, with increased activity sometimes manifest after 24 h of incubation. P. palmivora exhibited the lowest, and P. drechsleri exhibited the highest, overall peptidase activity. This fluorescent aminopeptidase profile procedure provides a relatively rapid method to supplement other taxonomic criteria for identification of these species of Phytophthora.