A Species-Specific Polymerase Chain Reaction Assay for Rapid Detection of Phytophthora nicotianae in Irrigation Water

ABSTRACT Phytophthora nicotianae is a common and destructive pathogen of numerous ornamental, agronomic, and horticultural crops such as tobacco, tomato, and citrus. We have developed a species-specific polymerase chain reaction (PCR) assay for rapid and accurate detection of this pathogen in irrigation water, a primary source of inoculum and an efficient means of propagule dissemination. This PCR assay consists of a pair of species-specific primers (PN), customization of a commercial soil DNA extraction kit for purification of DNA from propagules in irrigation water, and efficient PCR protocols for primer tests and sample detection. The PN primers proved adequately specific for P. nicotianae in evaluations with 131 isolates of P. nicotianae, 102 isolates from 15 other species of Phytophthora, and 64 isolates from a variety of other oomycetes, true fungi, and bacteria. These isolates originated from a wide range of host plants, three substrates (plant tissue, soil, and irrigation water), and numerous geographic locations. The detection sensitivity is between 80 and 800 fg DNA/mul. The assay detected the pathogen in naturally infested water samples from Virginia and South Carolina nurseries more rapidly and accurately than standard isolation methods. Use of this PCR assay can assist growers in making timely disease management decisions with confidence.     

利用基因YKT6鉴定疫霉属的不同种

 利用分子标记或对特异位点的碱基序列进行分析是植物病原物分子检测的基础,可以在属和种的水平上对物种进行区分和鉴定。对疫霉属的不同种已有一系列的分子检测方法。SNARE蛋白相关基因YKT6拥有保守的侧翼编码区,适于设计疫霉属特异性的PCR引物,同时其内含子所具有的多态性可开发出几乎所有疫霉种的分子标记。利用疫霉属特异性引物对P-YKT6-F/P-YKT6-R可在31个疫霉种中特异地扩增出一条约600 bp的条带,而在腐霉或其他真菌中不能扩增出该条带。利用大豆疫霉的引物对Ps-YKT6-F/ Ps-YKT6-R和辣椒疫霉的引物对Pc-YKT6-F/Pc-YKT6-R,能分别从大豆疫霉菌株和辣椒疫霉菌株中扩增出一条399 bp和282 bp的条带,常规PCR和巢式PCR的灵敏度分别达到100 pg和10 fg。利用这些引物也可从土壤和病组织中检测到目标病原菌。此外,利用上述特异性引物开发出了大豆疫霉和辣椒疫霉的实时定量PCR检测方法。基于YKT6基因的分子标记和检测方法可用于疫霉种的调查检测和法定定量检测。     

Diversity of Virulence in Phytophthora parasitica on Tobacco, as Reflected by Nuclear RFLPs

ABSTRACT A worldwide collection of P. parasitica isolates was investigated for the ability to infect tobacco and tomato, as related to elicitin production. Elicitin was produced by all nontobacco isolates, and nonproducing strains all were isolated from tobacco. In addition, producing strains were isolated from tobacco and coexisted with nonproducing (TE ) strains. Elicitin production generally was associated with low virulence on tobacco and frequent pathogenicity on tomato, whereas TE isolates generally were highly virulent and specialized to tobacco. Analysis of both mitochondrial and nuclear DNA restriction fragment length polymorphisms indicated, for the first time, that black shank isolates can be distinguished from other P. parasitica isolates on the basis of genetic criteria. Our results suggest that severe black shank is caused by a limited number of TE strains that have been disseminated by clonal evolution. Mutations in the TE phenotype seem to have arisen independently in several genetic backgrounds and distinct geographic areas. The fortuitous absence of elicitin production has precluded population replacements in areas of intensive tobacco cultivation. Thus, monitoring the loss of elicitin production in developing tobacco areas should be considered in disease management.     

拮抗细菌B8对烟草黑胫病菌的抑制作用及其菌株鉴定

从烟草根际土壤中分离到对烟草黑胫病菌及其它植物病原菌具有抑制作用的菌株B8,平板对峙培养抑菌带内菌丝畸形、原生质凝集或外渗。其发酵原液和发酵液的无菌滤液均能抑制烟草黑胫病菌和蜡状芽孢杆菌;离体叶片接种法测定其发酵液对烟草黑胫病的防效,结果表明预防作用达100%。室内盆栽试验表明B8菌株发酵液对烟草黑胫病的预防作用可达78.1%,优于治疗作用。该菌菌体杆状,芽孢侧生、椭圆形,经形态学、生理生化性状和16S rDNA序列测定,将其鉴定为侧孢短芽孢杆菌。     

Differential production of parasiticein, an elicitor of necrosis and resistance in tobacco, by isolates of Phytophthora parasitica

Fifteen isolates of Phytophthora parasitica , nine from tobacco (causing black shank disease) and six from other host plants, were compared by root inoculation with regard to their pathogenicity to young tobacco plants. A progressive invasion of the aerial parts over 1 week was observed only with the black shank isolates, while the non-tobacco isolates induced leaf necrosis within 2 days. Similar necrosis occurred when the roots of tobacco plants were dipped in diluted culture filtrates from non-tobacco isolates, but not in those from tobacco isolates. The necrosis-inducing filtrates were shown to contain a c. 10-kDa protein band which was not present in the other filtrates. This protein (named parasiticein) was purified by ion-exchange chromatography to homogeneity in SDS-PAGE and reverse-phase HPLC. Parasiticein was serologically related to cryptogein, a member of the elicitin family of proteinaceous elicitors previously described from other Phytophthora species. Like the other elicitins, parasiticein induced necrosis in tobacco plants and protected them against black shank. It most closely resembled capsicein in being acidic and in inducing resistance at concentrations (10–100 μg per plant) that caused little leaf necrosis. It is suggested that the absence of parasiticein production by the black shank isolates might be a factor involved in their specific pathogenicity to tobacco.     

Molecular Characterization of Natural Hybrids of Phytophthora nicotianae and P. cactorum

ABSTRACT Hybrid isolates of Phytophthora nicotianae x P. cactorum from five different hosts (Cyclamen, Lavandula, Lewisia, Primula, and Spathiphyllum spp.) were identified by their atypical morphology and their well-defined heterozygous isozyme patterns. The hybrid nature of these isolates was tested by restriction fragment length polymorphism analysis of the internal transcribed spacer (ITS) region of rDNA, generating fragments typical for both P. nicotianae and P. cactorum. In hybrid isolates, polymerase chain reactions (PCR) with primers derived from unique parts of the ITS region (ITS-PCR) of both species yielded a combination of unique amplicons typical of both parental species. Eleven hybrid isolates, three isolates of each parental species and two atypical isolates from Rhododendron and Idesia spp. close to P. cactorum, were analyzed for amplified fragment length polymorphisms (AFLP). Consistent differences in AFLP patterns existed among the hybrid isolates, strongly indicating that these hybrids have arisen from independent hybridization events between P. nicotianae and P. cactorum. The two atypical isolates morphologically resembling P. cactorum were identical to the latter species in ITS-restriction fragment length polymorphism and response to the specific PCR primers but were intermediate between P. nicotianae x P. cactorum and P. cactorum in isozyme profiles and AFLP patterns. Since the introduction of hydroponic systems in greenhouses in the Netherlands, outbreaks of Phytophthora diseases are occurring in previously unaffected host species. This may be due to interspecific hybridization events resulting in novel pathogenic behavior.     

Characterization of Phytophthora spp. Causing Outbreaks of Citrus Brown Rot in Florida

ABSTRACT Epidemics of citrus brown rot from 1994 to 1997 in the south-central and east-coast citrus areas of Florida were characterized and the causal Phytophthora spp. identified. Two species of Phytophthora, P. palmivora and P. nicotianae, were consistently associated with brown rot. Epidemics caused by P. palmivora appeared to be initiated on immature fruit dropped on the orchard floor. The soilborne fungus infected and sporulated on these fruit and was then disseminated to fruit above 1 m in the canopy. In contrast, infection by P. nicotianae, the common cause of root rot, was confined to the lowest 1 m of the canopy. Fruit infected by P. palmivora produced large amounts of ellipsoidal sporangia available for splash dispersal, whereas those infected by P. nicotianae produced far fewer spherical sporangia. Isolates from brown rot epidemics were compared with P. nicotianae from citrus in Florida and Texas, P. citrophthora in California, P. palmivora, and selected Phytophthora spp. from other hosts. Brown rot symptoms produced by the different pathogenic citrus isolates on inoculated fruit were indistinguishable. Morphology, mating behavior, and isozyme patterns of brown rot isolates from 1988 to 1997 matched P. palmivora from citrus roots, other host plants, and other locations, but were different from characterized isolates of P. citrophthora in California and P. nicotianae in Florida and Texas. Cellulose acetate electrophoresis of the isozyme glucose-6-phosphate isomerase rapidly identified the causal citrus pathogen from infected fruit and soil isolation plates. Although P. palmivora is an aggressive pathogen of citrus roots, bark, and fruit, populations in orchard soils were low compared with P. nicotianae.     

Loss of Production of the Elicitor Protein INF1 in the Clonal Lineage US-1 of Phytophthora infestans

ABSTRACT The extracellular protein INF1 of Phytophthora infestans is a member of the elicitin family of protein elicitors known to induce a hypersensitive response on some solanaceous and cruciferous plants. The presence of INF1 elicitin in culture filtrates of 102 P. infestans isolates from 15 countries was examined. All tested isolates produced INF1 except five isolates collected in 1976 and 1977 from infected potatoes in East Germany (the former German Democratic Republic). Based on hybridization to the multi-locus DNA fingerprint probe RG57, all the INF1-nonproducing isolates were shown to belong to the clonal lineage US-1 that dominated world populations until the 1980s. Phylogenetic analysis of a set of European US-1 isolates using amplified fragment length polymorphism fingerprint data indicated that loss of INF1 production evolved independently in separate lineages within US-1. DNA and RNA blot hybridizations showed that INF1-nonproducing isolates still retain a copy of the inf1 gene, whereas little inf1 mRNA could be detected. Hypothetical interpretations of the evolution in a restricted geographic area of P. infestans lineages deficient in the production of a specific elicitor protein are discussed.     

Elicitin诱导烟草微敏反应和防卫基因的表达

 研究了一种α型elicitin-parasiticein对烟草微敏反应(microscopic hypersensitive response, micro-HR)和防卫反应分子表征基因表达的诱导作用。用150 nmol/L的parasiticein喷洒烟草叶片12 h后, 经曲利本蓝(trypan blue)染色, 光镜下可观察到有细胞坏死, 说明parasiticein引起了micro-HR, 而水和低浓度的parasiticein不能引起micro-HR。用parasiticein注射叶片可引起肉眼可见的HR, 注射用parasiticein浓度远比喷洒引起micro-HR的浓度低。Parasiticein还可诱导对TMV的抗性, 浓度在30 nmol/L时比150 nmol/L的效果好。用parasiticein注射烟草30 min, HR表征基因hsr203J和hin1开始表达, 在9 h内逐渐降低, 12~16 h肉眼可观察到HR。Parasiticein喷洒烟草后, 病程相关蛋白(pathogenesis-related, PR)基因PR-1b在诱导的第1 d到第5 d都有一定量的表达。可见, parasiticein能同步诱导过敏反 应和抗病 性及其分 子表征基 因的表达 。     

Acquired resistance triggered by elicitins in tobacco and other plants

Elicitins are a family of proteins excreted byPhytophthora spp. They exhibit high sequence homology but large net charge differences. They induce necrosis in tobacco plants which then become resistant to the tobacco pathogenPhytophthora parasitica var.nicotianae. In stem-treated plants, resistance was not restricted to the site of elicitin application, but could be demonstrated by petiole inoculation at all levels on the stem. Resistance was already maximum after two days and lasted for at least two weeks. It was effective not only towardsP. p. var.nicotianae infection, but also against the unrelated pathogenSclerotinia sclerotiorum. In contrast to dichloroisonicotinic acid, an artificial inducer of systemic acquired resistance, which was increasingly effective with doses ranging from 0.25 to 5mole per plant, the basic elicitin cryptogein exhibited a threshold effect, inducing near total resistance and extensive leaf necrosis above 0.1 nmole per plant. Between 1 and 5 nmole, acidic elicitins (capsicein and parasiticein) protected tobacco plants with hardly any necrotic symptom. Elicitins exhibited similar effects in various tobacco cultivars andNicotiana species, although with quantitative differences, but induced neither necrosis nor protection in other SolanaceÆ (tomato, petunia and pepper). Among 24 additional species tested belonging to 18 botanical families, only some BrassicaceÆ, noticeably rape, exhibited symptoms in response to elicitins, in a cultivar-specific manner. Elicitins appear to be natural specific triggers for systemic acquired resistance and provide a tool for unraveling the mechanisms leading to its establishment.Abbreviations AR acquired resistance - HR hypersensitive response - INA 2,6-dichloroisonicotinic acid - Ppn Phytophthora parasitica var.nicotianae - SAR systemic acquired resistance     

荧光假单胞菌拮抗菌株对烟草疫霉的抑菌机制及控病效果

为探讨烟草根际生防细菌对烟草疫霉Phytophthora nicotianae的抑菌机制,从重庆地区连作烟田健康烟株根际土壤分离获得5株荧光假单胞菌Pseudomonas fluorescens拮抗菌株。通过平板对峙及代谢产物抑菌试验筛选对烟草疫霉具有高效拮抗作用的菌株,其中,P-72-10菌株抑菌效果最强,抑菌带半径达13.0 mm,相对抑制率为68.57%,且该菌株代谢产物对烟草疫霉菌丝生长有明显的抑制作用,相对抑制率达25.39%～46.03%;显微观察发现该菌株可引起烟草疫霉菌丝的分支增多,菌丝顶端膨大呈畸形,多数菌丝中间或顶端细胞的细胞壁加厚、原生质浓缩和产生类似厚壁孢子的细胞。在温室盆栽条件下P-72-10菌株对烟草黑胫病也表现出良好的控病效果,对抗病和感病品种的相对防效分别为53.57%和66.37%。     

Molecular Detection of Phytophthora ramorum by Real-Time Polymerase Chain Reaction Using TaqMan, SYBR Green, and Molecular Beacons

ABSTRACT Sudden oak death, caused by Phytophthora ramorum, is a severe disease that affects many species of trees and shrubs. This pathogen is spreading rapidly and quarantine measures are currently in place to prevent dissemination to areas that were previously free of the pathogen. Molecular assays that rapidly detect and identify P. ramorum frequently fail to reliably distinguish between P. ramorum and closely related species. To overcome this problem and to provide additional assays to increase confidence, internal transcribed spacer (ITS), beta-tubulin, and elicitin gene regions were sequenced and searched for polymorphisms in a collection of Phytophthora spp. Three different reporter technologies were compared: molecular beacons, TaqMan, and SYBR Green. The assays differentiated P. ramorum from the 65 species of Phytophthora tested. The assays developed were also used with DNA extracts from 48 infected and uninfected plant samples. All environmental samples from which P. ramorum was isolated by PARP-V8 were detected using all three real-time PCR assays. However, 24% of the samples yielded positive real-time PCR assays but no P. ramorum cultures, but sequence analysis of the coxI and II spacer region confirmed the presence of the pathogen in most samples. The assays based on detection of the ITS and elicitin regions using TaqMan tended to have lower cycle threshold values than those using beta-tubulin and seemed to be more sensitive.     

转隐地蛋白基因烟草的抗病性及遗传分析

 对转隐地蛋白(Cryptogein)基因烟草植株进行了抗病性测定、抗病分子机理及遗传分析的研究。结果表明:80%以上的转基因植株对烟草上3种重要病原菌的抗病性均增强,且能稳定遗传;转基因烟草植株的抗病性与整合的拷贝数成负相关,即绝大多数高度抗病的植株只含有1个拷贝,而感病植株一般为2~3个拷贝;部分转基因烟草植株Northern杂交分析表明:病程相关蛋白和渗透调节蛋白等防卫反应相关基因的表达丰度与转基因植株的抗病性存在着一定的正相关性,表达丰度越高,抗病性越强;综合农艺性状考察表明Cryptogein基因对烟草植株的生长有显著的促进作用。     

Molecular analysis of the major Phytophthora species on cocoa

The internally transcribed spacer (ITS) regions of the ribosomal RNA (rRNA) gene cluster of 161 isolates of Phytophthora species involved in pod rot, stem canker and leaf blight of cocoa were analysed to determine inter- and intraspecific variation in this disease complex. The species P. palmivora , P. megakarya , P. capsici , P. citrophthora and P. nicotianae could all be clearly distinguished by PCR amplification of the ITS region followed by restriction analysis with Hae III, Hinf I, Pvu II and Alu I. This method provided a relatively rapid identification procedure for these species, and was able to distinguish isolates that had previously been misidentified by morphological methods. Sequence analysis showed that the four main cocoa-associated species formed two distinct groups, one comprising P. capsici and P. citrophthora , and the other P. palmivora and P. megakarya . Detailed sequence analysis and comparison with published literature suggested that P. capsici isolates from cocoa may be closely related to P. tropicalis , a species recently described from Cyclamen and Dianthus .     

基于细胞色素氧化酶基因和激发素基因的大豆疫霉菌特异性PCR引物

引起大豆疫霉根腐病的大豆疫霉菌(Phytophthora sojae)是危害大豆的破坏性病原菌之一,也是我国重要的检疫性植物病原菌。简单、快速、准确的鉴定和检测技术是阻止大豆疫霉菌传入和病害早期诊断的有效工具。本研究从大豆疫霉菌细胞色素氧化酶基因Ⅱ(coxⅡ)序列和两个激发素(elicitin)家族基因EST序列中开发了3对大豆疫霉菌特异引物:Cox3-F/Cox3-R、PSEL1-F/PSEL1-R和PSEL2-F/PSEL2-R。这3对引物在大豆疫霉菌中分别扩增出450、289bp和370bp的特异性片段,其检测大豆疫霉菌基因组DNA的灵敏度分别为20、2pg/μL和2pg/μL。3对引物能够有效检测大豆疫霉菌侵染的大豆病株,可以用于病害诊断和鉴别。     

Mitochondrial haplotype analysis for differentiation of isolates of Phytophthora cinnamomi

Although Phytophthora cinnamomi is heterothallic, there are few instances of successful crossing in laboratory experiments, and analysis of field populations indicates a clonally reproducing population. In the absence of sexual recombination, the ability to monitor mitochondrial haplotypes may provide an additional tool for identification of clonal isolates and analysis of population structure. To determine mitochondrial haplotypes for this species, seven mitochondrial loci spanning a total of 6,961 bp were sequenced for 62 isolates representing a geographically diverse collection of isolates with A1 and A2 mating type. Three of the regions were primarily intergenic regions between trnG and rns, rns and nad3, and nad6 and cox1, while the remaining loci spanned cox2, nad9, rps10, and secY coding regions and some of the flanking spacer regions. In total, 45 mitochondrial haplotypes were identified (75% of the total isolates examined) with differences due to single-nucleotide polymorphisms (SNPs, totaling 152 bp) and length mutations (17 indels >2 bp representing a total of 910 bp in length). SNPs were the predominate mutation in the four coding regions and their flanking intergenic regions, while both SNPs and length mutations were observed in the three primarily intergenic regions. Some of the length mutations in these regions were due to addition or loss of unique sequences while others were due to variable numbers of subrepeats (in the trnG-rns region, there were 3 to 12 copies of a 24-bp subrepeat sequence that differentiated 17 haplotypes). Network analysis of the haplotypes identified eight primary clades, with the most divergent clade representing primarily A1 isolates collected from Papua New Guinea. The isolate grouping in the network corresponded to mating type and previously published isozyme classifications, with three exceptions: a haplotype representing an A1 mating type (H29) was placed well within the A2 mating type haplotype grouping, one haplotype (H26) had isolates with two isozyme classifications, and one isozyme group was represented on separate network clades, suggesting that recombination has occurred in the past. Among the 62 isolates examined, several examples were identified of isolates recovered from different geographic regions having the same mitochondrial haplotype, suggesting movement of isolates via plant material. Analysis of the data set to determine whether fewer loci could be sequenced to classify haplotypes indicated that the trnG-rns and rns-nad6 loci would classify 87% of the haplotypes identified in this study, while additional sequencing of the nad9 or secY loci would further differentiate the remaining six haplotypes. Based on conservation of gene order in Phytophthora spp., the trnG-rns locus should be useful for mitochondrial haplotype classification in other species, as should the cox2, nad9, rps10, and secY loci. However, the rns-nad3 and nad6-cox1 loci span regions that can have a different gene order in some Phytophthora spp.     

Cytological Characterization of Elicitin-Induced Protection in Tobacco Plants Infected by Phytophthora parasitica or Phytoplasma

ABSTRACT Elicitins, small proteins secreted by Phytophthora and Pythium spp., display the ability to induce plant resistance toward pathogens. Ultrastructural investigations of cryptogein-treated tobacco plants evidenced host defense responses such as (i) formation of a calcium pectate gel in intercellular spaces of parenchymas, (ii) impregnation of pectin by phenolic compounds in intercellular spaces of phloem bundles, and (iii) accumulation of phloem proteins (P proteins) in the lumen of leaf sieve elements. These cytological modifications lead to the enhancement of physical barriers that prevent pathogen ingress and restrict host tissue colonization when cryptogein-treated tobacco plants were challenged with the pathogen Phytophthora parasitica. Wall appositions also were observed at most sites of penetration of hyphae. Moreover, growing hyphae exhibited severe morphological damages, suggesting a modified toxic environment. The same induction of P proteins in mature sieve tubes of tobacco leaves was obtained with oligandrin treatment, another elicitin. Cryptogein or oligandrin treatment prevented symptom expression in phytoplasma-infected tobacco plants in contrast with nontreated tobacco plants. Moreover, P protein plugs and occlusion of pore sites by callose were evidenced in sieve elements of treated plants. Both these phloem modifications might prevent the in planta movement of phloem-restricted microorganisms.     

Pathogenicity and RAPD analysis of Phytophthora nicotianae pathogenic to pepper in Tunisia

Nine isolates of Phtophthora nicotianae were isolated from infected pepper plants. Their pathogenicity was studied in Capsicum annuum in comparison with P. nicotianae isolates from tomato and tobacco. The pathogenicity test showed that pepper isolates of P. nicotianae are adapted to their host. Banding patterns obtained by RAPD analysis with six oligonucleotide primers revealed polymorphism that grouped the isolates independently of the plant host. The polygenic dendrogram showed that pepper isolates were more similar to tomato isolates than to tobacco isolates. The RAPD bands of 1300 and 1500 bp, detected with primers OPD-01 and OPD-10, respectively, appeared specific to the most pathogenic pepper isolates. The OPK-08-1950 seems specific to the isolates of P. nicotianae from tomato. These results suggest that host specified might occur in P. nicotianae and that may be due to interspecific hybridization events resulting in novel pathogenic behavior.     

Protection of Citrus Rootstocks Against Phytophthora spp. with a Hypovirulent Isolate of Phytophthora nicotianae

ABSTRACT Phytophthora root rot of citrus in Florida is caused by Phytophthora nicotianae and P. palmivora. A naturally occurring isolate of P. nicotianae (Pn117) was characterized as hypovirulent on citrus roots. Pn117 infected and colonized fibrous roots, but caused significantly less disease than the virulent isolates P. nicotianae Pn198 and P. palmivora Pp99. Coincident inoculation of rootstock seedlings of Cleopatra mandarin (Citrus reticulata) or Swingle citrumelo (C. paradisi x Poncirus trifoliata) with the hypovirulent Pn117 and the virulent isolates Pn198 and Pp99 did not reduce the severity of disease caused by the virulent Phytophthora spp. When either rootstock was inoculated with the hypovirulent Pn117 for 3 days prior to inoculation with virulent isolates, preinoculated seedlings had significantly less disease and greater root weight compared with seedlings inoculated with the virulent isolates alone. Recovery of the different colony types of Phytophthora spp. from roots of sweet orange (C. sinensis) or Swingle citrumelo was evaluated on semiselective medium after sequential inoculations with the hypovirulent Pn117 and virulent Pp99. Pn117 was isolated from roots at the same level as the Pp99 at 3 days post inoculation. Preinoculation of Pn117 for 3 days followed by inoculation with Pp99 resulted in greater recovery of the hypovirulent isolate and lower recovery of the virulent compared with coincident inoculation.