Occurrence and Identification of Phytophthora spp. Pathogenic to Pear Fruit in Irrigation Water in the Wenatchee River Valley of Washington State

ABSTRACT Seven hundred forty-nine isolates of Phytophthora spp. were obtained from irrigation canals in eastern Washington State during the 1992 to 1995 and 1999 growing seasons. Isolates were retrieved using pear baiting techniques. All isolates were pathogenic to pear and were present in irrigation water beginning early in fruit development. Over the course of the 5 year study, 10 and 5% of isolates were identified as P. cactorum and P. citricola, respectively, using morphological criteria. The remaining isolates could not be identified using morphological criteria. Colony morphology of these isolates was characterized during all years of the study. In 1999, more detailed studies utilizing polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) analysis of entire internal transcribed spacer (ITS) regions (ITS1, 5.8S, and ITS2) of ribosomal DNA for 180 isolates, and sequence analysis of ITS2 for 50 isolates, were used to investigate genetic variation and phylogenetic relationships among isolates. Isolates were divided into 12 groups based on their growth type on corn meal agar. Restriction digestion of the entire ITS region with three enzymes revealed 11 restriction digestion patterns among 180 isolates. PCR-RFLP and sequence data were obtained for 12 reference Phytophthora spp. (two species in each of Waterhouse's six morphological groups). Phylogenetic analysis of ITS2 regions revealed nine clades, each with strong bootstrap support. Molecular analyses revealed 23 isolates that were in the P. gonapodyides clade, 9 in the P. parasitica clade, 1 in the P. cactorum clade, 7 in the P. citricola/capsici clade, and 4 in the P. cambivora/pseudotsugae clade. The three isolates comprising clade 5 were significantly distinct from all other Phytophthora spp. in the databases and may represent a new Phytophthora sp. Colony morphology was not consistently correlated to PCR-RFLP pattern or ITS2 phylogeny, suggesting that the former criterion is insufficient for species identification. The results of this study indicate that at least nine phylogenetically distinct taxa of Phytophthora pathogenic to pear are present in irrigation water in North Central Washington.     

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.     

双重PCR检测马铃薯晚疫病菌和青枯病菌方法的建立及应用

 利用真菌通用引物ITS1和ITS4扩增马铃薯晚疫病菌转录间隔区并进行序列测定,通过序列比较,设计了1对马铃薯晚疫病菌的特异引物INF1/INF2,并对15种不同真菌、细菌和7种疫霉属和腐霉属卵菌基因组DNA进行PCR扩增,结果只有不同来源的马铃薯晚疫病菌株可获得324 bp的特异带。将引物INF1/INF2与卵菌通用引物进行巢式PCR扩增后,其检测灵敏度在DNA水平上可达30 fg。运用设计的引物与马铃薯青枯病菌特异引物结合建立了双重PCR体系,能从马铃薯晚疫病菌和马铃薯青枯病菌总基因组DNA以及人工接种和自然发病的马铃薯植株中分别或同时扩增到324 bp和281 bp的特异片段。实现了同时对马铃薯晚疫病菌和马铃薯青枯病菌的快速可靠检测。     

Genetic and Morphological Diversity of Temperate and Tropical Isolates of Phytophthora capsici

ABSTRACT Phytophthora capsici is a diverse species causing disease on a broad range of both temperate and tropical plants. In this study, we used cultural characteristics, amplified fragment length polymorphism (AFLP), and DNA sequence analyses of the ribosomal internal transcribed spacer (ITS) region and mitochondrial cytochrome oxidase II (cox II) genes to characterize temperate and tropical isolates from a wide range of host species. All but one temperate isolate grew at 35 degrees C, while all tropical isolates did not. All but two tropical isolates formed chlamydospores, while temperate isolates did not. There was strong bootstrap support for separation of temperate and tropical isolates using AFLP analysis; however, the temperate isolates appeared as a subgroup within the observed variation of the tropical isolates. The majority of temperate isolates clustered within a single clade with low variation regardless of host or geographical origin, while the tropical isolates were more variable and grouped into three distinct clades. Two clades of tropical isolates grouped together and were affiliated closely with the temperate isolates, while the third tropical clade was more distantly related. Phylogenetic analysis of the ITS regions resulted in similar groupings and variation within and between the temperate and tropical isolates as with the AFLP results. Sequence divergence among isolates and clades was low, with more variation within the tropical isolates than within the temperate isolates. Analysis of other species revealed shorter branch lengths separating temperate and tropical isolates than were observed in comparisons among other phylogenetically closely related species in the genus. Analysis of cox II sequence data was less clear. Although the temperate and tropical isolates grouped together apart from other species, there was no bootstrap support for separating these isolates. Restriction fragment length polymorphism (RFLP) analysis of the ITS regions separated the temperate and tropical isolates, as in the AFLP and ITS phylogenetic analyses. However, RFLP analysis of the cox I and II gene cluster did not distinguish between temperate and tropical isolates. The differences in grouping of isolates in these two RFLP studies should be helpful in identifying isolate subgroups. Our data do not fully clarify whether or not temperate and tropical isolates should be separated into different species. The available worldwide data are incomplete and the full range of variation in the species is not yet known. We suggest refraining from using the epithet P. tropicalis until more data are available.     

Genetic variation among asexual progeny of Phytophthora infestans detected with RAPD and AFLP markers

Genotypic variation among 32 single-zoospore isolates (SZI) of Phytophthora infestans , derived asexually from two hyphal-tip parental isolates (PI-105 and PI-1) of the US-8 genotype, was assessed with 80 random amplified polymorphic DNA (RAPD) primers and 18 amplified fragment length polymorphic DNA (AFLP) primer pairs. In previous investigations, the SZIs from parental isolate PI-105 showed high levels of virulence variability and were differentiated into 14 races, whereas the SZIs from PI-1 showed identical virulence to the parent. The purpose of this investigation was to determine if phenotypic variation observed among SZIs of P. infestans could be detected at the DNA level in these isolates. Polymorphism was detected with 51 RAPD primers and with all 18 AFLP primer pairs in PI-105 SZIs. In SZIs from PI-1, polymorphism was also detected with 25 RAPD primers and 17 AFLP primer pairs. Cluster analysis using the unweighted pair-group method with arithmetic averages (UPGMA) separated the SZIs from parent PI-105 into six virulence groups, 11 RAPD groups and three AFLP groups. Cluster analysis of PI-1 SZIs, which all belong to the same virulence group, differentiated them into four RAPD groups and six AFLP groups. No close correlation among RAPD, AFLP and virulence groups could be established within the two progenies of SZIs. Results of this study suggest that there is a considerable level of inherent genetic variability among SZIs derived asexually from the same parental isolate. The possible mechanisms and implications of this genetic variation are discussed.     

Detection of <Emphasis Type="Italic">Phytophthora nicotianae</Emphasis> and <Emphasis Type="Italic">P. palmivora</Emphasis> in citrus roots using PCR-RFLP in comparison with other methods

Phytophthora nicotianae and P. palmivora are the most important soil-borne pathogens of citrus in Florida. These two species were detected and identified in singly and doubly infected plants using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) of internal transcribed spacer (ITS) regions of ribosomal DNA. The sensitivity of the PCR-RFLP was analyzed and the usefulness of the method evaluated as an alternative or supplement to serological methods and recovery on semi-selective medium. In a semi-nested PCR with universal primers ITS4 and ITS6, the detection limit was 1 fg of fungal DNA, which made it 1000× more sensitive than a single-step PCR with primers ITS4 and DC6. The sensitivity of detection for P. nicotianae was shown to be ten-fold lower than for P. palmivora, limiting its detection with restriction profiles in plants infected by both fungal species. Phytophthora nicotianae was detected with species-specific primers in all samples inoculated with this species despite the absence of species-specific patterns in RFLP. In contrast, the incidence of detection of P. palmivora in the presence of P. nicotianae was considerably lower using plating and morphological detection methods. Due to its high sensitivity, PCR amplification of ribosomal ITS regions is a valuable tool for detecting and identifying Phytophthora spp. in citrus roots, provided a thorough knowledge of reaction conditions for the target species is established prior to the interpretation of data.     

新疆主要农作物疫霉菌种类鉴定

 1993~1995年,对侵染新疆主要农作物的疫霉菌进行了较全面的调查研究,共从9个地区的28种作物上采集表现根腐、根颈腐、果腐症状(包括病土)的病样1531个,结果从其中17种作物上分离到452个疫霉菌株。根据形态特征、生理生化特性、致病性和菌体可溶性蛋白电泳测定,鉴定为7个种:辣椒疫霉(Phytophthora capsici Leon.),恶疫霉[P.cactorum(Leb.et Cohn) Schroeter],掘氏疫霉(P.drechsleri Tucker),菸草疫霉(P.nicotianae van Breda de Haan),苎麻疫霉(P.boehm eriaeSaw.),柑桔褐腐疫霉[P.citrophthora (Sm.et Sm.) Leonian],隐地疫霉(P.cryptogea Pethyb.etLaff.)。这些疫菌是造成新疆茄果类、瓜类、棉花、苹果、梨、枸杞、草霉、红花、白术等幼苗及成株大量死亡及烂果的主要病原,在农业生产中具有十分重要的意义。     

Specific detection of Phytophthora cactorum in diseased strawberry plants using nested polymerase chain reaction

Validated protocols for DNA purification and PCR amplification are reported for detection of Phytophthora cactorum in diseased strawberry plants. To remove PCR inhibitors, necrotic strawberry tissues were soaked in 5% alconox solution for >12 h before DNA extraction, and the extracted genomic DNA was embedded in an agarose gel chamber and subjected to electrophoresis. The purified DNA was amplified reliably by PCR. Nested PCR was used to detect a portion of the rRNA gene of P. cactorum in samples. In the first round of PCR, primers ITS1 and ITS4 amplified fragments of varying sizes from total genomic DNA from diseased strawberry plants. In the second round of PCR, a 1:25 dilution of the first-round PCR products was used as template with two P. cactorum- specific primer pairs (BPhycacL87FRG and BPhycacR87RRG, which amplified a 340-bp fragment and a 480-bp fragment from the rRNA gene; and BPhycacL89FRG and BPhycacR176RRG, which amplified a 431-bp fragment). Validation tests using culture-based isolations as a standard for comparison indicated that the DNA purification and PCR primers and amplification protocols were reliable and specifically amplified a portion of the rRNA gene of P. cactorum from necrotic root, crown and petiole tissues of strawberry naturally infected by the pathogen.     

Standard and Swedish variant types of the hybrid alder Phytophthora attacking alder in Hungary

A new Phytophthora disease of common alder (Alnus glutinosa) similar to that previously reported in several countries in Europe has been observed in Hungary. Based on these earlier studies, the alder Phytophthora was considered likely to be a hybrid between P cambivora and a P fragariae-like species: across Europe a range of new alder Phytophthora is spreading that comprise a range of heteroploid hybrids including a 'standard' hybrid type and several other hybrid types termed 'variants'. Phenotypic and molecular features of the pathogen in Hungary were characterised and compared with isolates from elsewhere. The morphologies of five isolates from one region (Hévíz) resembled the common, 'standard' type, whereas the three isolates from another region (Hanság) exhibited traits similar to those of one of the 'variant' types, ie the Swedish 'variant'. Molecular markers of these two groups of Hungarian isolates also represented a good fit to those of the standard type and the Swedish variant, respectively. Isozyme patterns and profiles of restriction fragments of the entire internal transcribed spacer (ITS) region or mitochondrial DNAs and of RAPD-PCR products did not differ within a group, but distinct polymorphisms were exhibited between the two groups of isolates. Southern analysis of random amplified polymorphic DNA (RAPD) revealed the homologous nature of co-migrating bands of P cambivora and the isolates of alder Phytophthora. Furthermore, restriction fragment profiles of the ITS region of ribosomal DNAs and the mtDNAs were consistent with reported biparental origin of alder Phytophthora. The hybrid status of these continuously evolving pathogens raises many issues and challenges concerning efficient control measures.     

Assessment of Diversity in Claviceps africana and Other Claviceps Species by RAM and AFLP Analyses

ABSTRACT Genetic diversity among isolates of Claviceps africana, the sorghum ergot pathogen, and isolates of other Claviceps spp. causing ergot on sorghum or other hosts, was analyzed by random amplified microsatellite (RAM) and amplified fragment length polymorphism (AFLP) analyses. Of the RAM primer sets tested, one revealed polymorphism in C. africana isolates, with Australian and Indian isolates possessing a unique fragment. AFLP analysis, in addition to clearly distinguishing Claviceps spp., revealed polymorphisms in C. africana. A group of isolates from the United States, Puerto Rico, and South Africa exhibited 95 to 100% similarity with one another. Several isolates from Isabela, Puerto Rico were 100% similar to an isolate from Texas, and another isolate from Puerto Rico was identical with one from Nebraska. Australian and Indian isolates showed greater than 90% similarity with isolates from the United States., Puerto Rico, and South Africa. A number of polymorphisms existed in the United States group, indicating that the recently introduced population contains multiple genotypes. Isolates of C. sorghicola, a newly described sorghum pathogen from Japan, were very distinct from other species via RAM and AFLP analyses, as were isolates from outgroups C. purpurea and C. fusiformis. Both RAM and AFLP analysis will be useful in determining future patterns of intercontinental migration of the sorghum ergot pathogen, with the AFLP method showing greater ability to characterize levels of intraspecific variation.     

The Population Structure of Phytophthora infestans from the Toluca Valley of Central Mexico Suggests Genetic Differentiation Between Populations from Cultivated Potato and Wild Solanum spp

ABSTRACT The population structure of Phytophthora infestans in the Toluca Valley of central Mexico was assessed using 170 isolates collected from cultivated potatoes and the native wild Solanum spp., S. demissum and S. xendinense. All isolates were analyzed for mitochondrial DNA (mtDNA) haplotype and amplified fragment length polymorphism (AFLP) multi-locus fingerprint genotype. Isolate samples were monomorphic for mtDNA haplotype because all isolates tested were of the Ia haplotype. A total of 158 multilocus AFLP genotypes were identified among the 170 P. infestans isolates included in this study. P. infestans populations sampled in the Toluca Valley in 1997 were highly variable and almost every single isolate represented a unique genotype based on the analysis of 165 AFLP marker loci. Populations of P. infestans collected from the commercial potato-growing region in the valley, the subsistence potato production area along the slopes of the Nevado de Toluca, and the native Solanum spp. on the forested slopes of the volcano showed a high degree of genetic diversity. The number of polymorphic loci varied from 20.0 to 62.4% for isolates collected from the field station and wild Solanum spp. On average, 81.8% (135) of the AFLP loci were polymorphic. Hetero-zygosity varied between 7.7 and 19.4%. Significant differentiation was found at the population level between strains originating from cultivated potatoes and wild Solanum spp. (P = 0.001 to 0.022). Private alleles were observed in individual isolates collected from all three populations, with numbers of unique dominant alleles varying from 9 to 16 for isolates collected from commercial potato crops and native Solanum spp., respectively. Four AFLP markers were exclusively found present in isolates collected from S. demissum. Indirect estimation of gene flow between populations indicated restricted gene flow between both P. infestans populations from cultivated potatoes and wild Solanum hosts. There was no evidence found for the presence of substructuring at the subpopulation (field) level. We hypothesize that population differentiation and genetic isolation of P. infestans in the Toluca Valley is driven by host-specific factors (i.e., R-genes) widely distributed in wild Solanum spp. and random genetic drift.     

Development and application of a PCR-based 'molecular tool box' for the identification of Phytophthora species damaging forests and natural ecosystems

A PCR-based 'molecular tool box', based on a region of the ras-related protein gene Ypt 1, was developed for the identification of 15 Phytophthora species that damage forests and trees: P. cactorum , P. cambivora , P. cinnamomi , P. citricola , P. europaea , P. inundata , P. lateralis , P. megasperma , P. nemorosa , P. kernoviae , P. pseudosyringae , P. psychrophila , P. quercina , P. ramorum and P. ilicis . Most primers proved highly specific in blast analyses and in tests with DNA from 72 isolates of 35 species of Phytophthora and nine species representative of Pythium . Exceptions were primers designed for P. cactorum and P. ilicis , which cross-reacted with P. idaei and P. nemorosa , respectively. Amplification with Phytophthora -genus-specific primers before amplification with the various species-specific primers (nested PCR) increased the sensitivity of detection over amplification with species-specific primers only: detection limits ranged between 100 and 10 pg target DNA µ L⁻¹ in the latter, compared with 100 fg µ L⁻¹ in nested PCR. Using existing methods for rapid extraction and purification of DNA, single-round amplification was appropriate for detection of target Phytophthora species in leaves, but nested PCR was required for soil and water samples. The quarantine pathogens P. ramorum and P. kernoviae were detected in a number of naturally infected leaves collected in England and Wales, whereas P. citricola was commonest in water and soil samples from natural Scottish ecosystems.     

Identification of some oomycetes by reverse dot blot hybridization

ABSTRACT An assay was developed that can identify unknown isolates of Pythium or Phytophthora species in a single hybridization. This reverse dot blot system is based on arrays of species-specific amplified fragments or oligonucleotides derived from the internal transcribed spacer (ITS) region, which are blotted as dots on a nylon membrane. By using total DNA from a sample as the template, universal primers, and digoxigenin-dUTP, the ITS was amplified and labeled simultaneously by the polymerase chain reaction (PCR). A small aliquot of the resultant labeled and amplified product was used as a probe for hybridization to a dot blot membrane that contained the immobilized species-specific oligonucleotides or amplified PCR fragments. The reverse dot blot system based on arrays of oligonucleotides showed far fewer cross-hybridizations than one based on entire amplified ITS I fragments. Unknown species can be identified simply by visualizing the positive hybridization reaction between the DNA labeled directly from the sample and the immobilized specific oligonucleotide. Currently, the assay can be used to identify Pythium aphanidermatum, P. ultimum, P. acanthicum, and Phytophthora cinnamomi. An oligonucleotide that was originally designed to identify Phytophthora hybridized to 10 of the 14 Phytophthora species tested. Another oligonucleotide designed to identify oomycetes hybridized to the 68 species tested, which represented two of the four orders of this phylum.     

Oidium neolycopersici: intraspecific variability inferred from amplified fragment length polymorphism analysis and relationship with closely related powdery mildew fungi infecting various plant species

Previous works indicated a considerable variation in the pathogenicity, virulence, and host range of Oidium neolycopersici isolates causing tomato powdery mildew epidemics in many parts of the world. In this study, rDNA internal transcribed spacer (ITS) sequences, and amplified fragment length polymorphism (AFLP) patterns were analyzed in 17 O. neolycopersici samples collected in Europe, North America, and Japan, including those which overcame some of the tomato major resistance genes. The ITS sequences were identical in all 10 samples tested and were also identical to ITS sequences of eight previously studied O. neolycopersici specimens. The AFLP analysis revealed a high genetic diversity in O. neolycopersici and indicated that all 17 samples represented different genotypes. This might suggest the existence of either a yet unrevealed sexual reproduction or other genetic mechanisms that maintain a high genetic variability in O. neolycopersici. No clear correlation was found between the virulence and the AFLP patterns of the O. neolycopersici isolates studied. The relationship between O. neolycopersici and powdery mildew anamorphs infecting Aquilegia vulgaris, Chelidonium majus, Passiflora caerulea, and Sedum alboroseum was also investigated. These anamorphs are morphologically indistinguishable from and phylogenetically closely related to O. neolycopersici. The cross-inoculation tests and the analyses of ITS sequences and AFLP patterns jointly indicated that the powdery mildew anamorphs collected from the above mentioned plant species all represent distinct, but closely related species according to the phylogenetic species recognition. All these species were pathogenic only to their original host plant species, except O. neolycopersici which infected S. alboroseum, tobacco, petunia, and Arabidopsis thaliana, in addition to tomato, in cross-inoculation tests. This is the first genome-wide study that investigates the relationships among powdery mildews that are closely related based on ITS sequences and morphology. The results indicate that morphologically indistinguishable powdery mildews that differed in only one to five single nucleotide positions in their ITS region are to be considered as different taxa with distinct host ranges.     

胶孢炭疽菌及两种疫霉菌对苯酰菌胺的敏感性与

选择对多菌灵、乙霉威和苯酰菌胺具有不同敏感性的胶孢炭疽菌Colletotrichum gloeosporioides、辣椒疫霉菌Phytophthora capsici及恶疫霉菌P. cactorum,分析其β-微管蛋白氨基酸突变与敏感性的关系。结果表明,胶孢炭疽菌对苯酰菌胺、多菌灵和乙霉威的敏感性与β-微管蛋白198位或200位氨基酸突变有关：对多菌灵敏感,对苯酰菌胺和乙霉威不敏感的胶孢炭疽菌β-微管蛋白氨基酸198位为谷氨酸（E）,200位为苯丙氨酸（F）;对多菌灵已产生抗性而对苯酰菌胺和乙霉威不敏感的菌株,其氨基酸200位由苯丙氨酸（F）突变为了酪氨酸（Y）;对多菌灵高抗,对苯酰菌胺和乙霉威敏感的菌株其氨基酸198位由谷氨酸（E）突变为了丙氨酸（A）。辣椒疫霉菌和恶疫霉菌对苯酰菌胺敏感,对多菌灵和乙霉威均不敏感。检测疫霉菌菌株β-微管蛋白未发现氨基酸突变,但发现其β-微管蛋白氨基酸在196~200位与胶孢炭疽菌差异较大,这可能是导致苯酰菌胺仅对疫霉菌有抑制效果的原因。     

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.     

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.     

Cross-reactivity of antiserum raised against Phytophthora fragariae with other Phytophthora species and its evaluation as a genus-detecting antiserum

Antiserum (anti-PfM) raised against mycelial suspensions of Phytophthora fragariae isolates reacted strongly with antigens from several Phytophthora species. Some cross-reactions with antigens from Pythium species were decreased by fractionating on an affinity column of Sepharose 4B bound to extracts of Fragaria vesca roots infected with P. fragariae. The affinity-purified anti-PfM retained its high cross-reactivity with the various Phytophthora species tested. It also detected infection of raspberry and strawberry roots by some Phytophthora species. This antiserum could, therefore, prove useful as a broad-spectrum Phytophthora-detecting antiserum.
Anti-PfM could not be made specific for P. fragariae because it was raised against components shown to be antigenically similar in all Phytophthora species tested. However, immunoblotting with the affinity-purified anti-PfM produced distinct patterns for P. fragariae, P. erythroseptica and P. cactorum: three serotypes were identified for the latter species. This antiserum might therefore prove useful in classifying Phytophthora species.     

Specific and Sensitive Detection of Phytophthora nicotianae By Simple and Nested-PCR

Phytophthora nicotianae Breda de Haan is one of the most important soil-borne plant pathogens. The identification of this pathogen based on morphological or physiological characters is time-consuming and labour-intensive and requires comprehensive knowledge of fungi. Molecular analysis of the internal transcribed spacer (ITS) regions of rDNA is a novel and very effective method of species determination. Based on this concept, conventional and single closed tube nested-PCRs were developed for the specific and sensitive detection of P. nicotianae. Two new specific primers, designed from the spacer regions ITS1 and ITS2, internal to the nucleotide sequence flanked by universal primers ITS4 and ITS6, were used. To evaluate the specificity of the method, 36 morphologically characterized isolates were tested. A positive reaction, characterized by an amplification product of 737 bp, was shown by all P. nicotianae isolates and two P. nicotianae/cactorum hybrids. No amplification product was observed when other Phytophthora species and genera were assayed. The sensitivity of this method was analysed by serial dilutions of a defined amount of fungal DNA in a healthy root extract. Nested-PCR was at least 1000 times more sensitive than conventional PCR. In addition, samples from different infection sites, origins and crops, samples from nutrient solution, water and the rockwool used in hydroponic cultures, were analysed to validate this method.