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Unlabelled: Phytophthora species were isolated from rivers and streams in the southwestern United States by leaf baiting and identified by sequence analysis of internal transcribed spacer (ITS) ribosomal DNA (rDNA). The major waterways examined included the Rio Grande River, Gila River, Colorado River, and San Juan River. The most prevalent species identified in rivers and streams were Phytophthora lacustris and P. riparia, both members of Phytophthora ITS clade 6. P. gonapodyides, P. cinnamomi, and an uncharacterized Phytophthora species in clade 9 were also recovered. In addition, six isolates recovered from the Rio Grande River were shown to be hybrids of P. lacustris × P. riparia Pathogenicity assays using P. riparia and P. lacustris failed to produce any disease symptoms on commonly grown crops in the southwestern United States. Inoculation of Capsicum annuum with P. riparia was shown to inhibit disease symptom development when subsequently challenged with P. capsici, a pathogenic Phytophthora species., Importance: Many Phytophthora species are significant plant pathogens causing disease on a large variety of crops worldwide. Closer examinations of streams, rivers, and forest soils have also identified numerous Phytophthora species that do not appear to be phytopathogens and likely act as early saprophytes in aquatic and saturated environments. To date, the Phytophthora species composition in rivers and streams of the southwestern United States has not been evaluated. This article details a study to determine the identity and prevalence of Phytophthora species in rivers and streams located in New Mexico, Arizona, Colorado, Utah, and Texas. Isolated species were evaluated for pathogenicity on crop plants and for their potential to act as biological control agents., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

Oomycetes from the genus Phytophthora are among the most important plant pathogens in agriculture. Epidemics caused by P. infestans precipitated the great Irish famine and had a major impact on society and human history. In the tropics, P. palmivora is a pathogen of many plant species including cacao (Theobroma cacao), citrus (Citrus sp.), durian (Durio zibethines), jackfruit (Artrocarpus heterophyllus), rubber (Hevea brasiliensis), and several palm species including coconut (Cocos nucifera), and the African oil palm (Elaeis guineensis) as determined recently. The first localized epidemics of bud rot in oil palm in Colombia were reported in 1964. However, recent epidemics of bud rot have destroyed more than 70,000 ha of oil palm in the Western and Central oil palm growing regions of Colombia. The agricultural, social, and economic implications of these outbreaks have been significant in Colombia. Identification of the pathogen after 100 years of investigating the disease in the world enabled further understanding of infection, expression of a range of symptoms, and epidemiology of the disease. This review examines the identification of P. palmivora as the cause of bud rot in Colombia, its epidemiology, and discusses the importance of P. palmivora as a major threat to oil palm plantings globally.

Background: Currently, real-time polymerase chain reaction (qPCR) is the technique most often used to quantify pathogen presence. Digital PCR (dPCR) is a new technique with the potential to have a substantial impact on plant pathology research owing to its reproducibility, sensitivity and low susceptibility to inhibitors. In this study, we evaluated the feasibility of using dPCR and qPCR to quantify Phytophthora nicotianae in several background matrices, including host tissues (stems and roots) and soil samples., Results: In spite of the low dynamic range of dPCR (3 logs compared with 7 logs for qPCR), this technique proved to have very high precision applicable at very low copy numbers. The dPCR was able to detect accurately the pathogen in all type of samples in a broad concentration range. Moreover, dPCR seems to be less susceptible to inhibitors than qPCR in plant samples. Linear regression analysis showed a high correlation between the results obtained with the two techniques in soil, stem and root samples, with R(2) = 0.873, 0.999 and 0.995 respectively., Conclusions: These results suggest that dPCR is a promising alternative for quantifying soil-borne pathogens in environmental samples, even in early stages of the disease., (© 2015 Society of Chemical Industry.)

In this study, we report on a novel approach for the label-free and species-specific detection of the plant pathogen Phytophthora ramorum from real samples using surface enhanced Raman scattering (SERS). In this context, we consider the entire analysis chain including sample preparation, DNA isolation, amplification and hybridization on SERS substrate-immobilized adenine-free capture probes. Thus, the SERS-based detection of target DNA is verified by the strong spectral feature of adenine which indicates the presence of hybridized target DNA. This property was realized by replacing adenine moieties in the species-specific capture probes with 2-aminopurine. In the case of the matching capture and target sequence, the characteristic adenine peak serves as an indicator for specific DNA hybridization. Altogether, this is the first assay demonstrating the detection of a plant pathogen from an infected plant material by label-free SERS employing DNA hybridization on planar SERS substrates consisting of silver nanoparticles.

Phytophthora capsici is a virulent oomycete pathogen of many vegetable crops. Recently, it has been demonstrated that the recognition of the RXLR effector AVR3a1 of P. capsici (PcAVR3a1) triggers a hypersensitive response and plays a critical role in mediating non-host resistance. Here, we analyzed the occurrence of PcAVR3a1 in 57 isolates of P. capsici derived from globe squash, eggplant, tomato and bell pepper cocultivated in a small geographical area. The occurrence of PcAVR3a1 in environmental strains of P. capsici was confirmed by PCR in only 21 of these pathogen isolates. To understand the presence-absence pattern of PcAVR3a1 in environmental strains, the flanking region of this gene was sequenced. PcAVR3a1 was found within a genetic element that we named PcAVR3a1-GI (PcAVR3a1 genomic island). PcAVR3a1-GI was flanked by a 22-bp direct repeat, which is related to its site-specific recombination site. In addition to the PcAVR3a1 gene, PcAVR3a1-GI also encoded a phage integrase probably associated with the excision and integration of this mobile element. Exposure to plant induced the presence of an episomal circular intermediate of PcAVR3a1-GI, indicating that this mobile element is functional. Collectively, these findings provide evidence of PcAVR3a1 evolution via mobile elements in environmental strains of Phytophthora., (© FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

A rapid and simple instrument-free detection system was developed for the identification of the plant pathogen Phytophthora kernoviae (P. kernoviae). The on-site operable analysis steps include magnetic particle based DNA isolation, helicase-dependent amplification (HDA) and chip-based DNA hybridization. The isothermal approach enabled the convenient amplification of the yeast GTP-binding protein (Ypt1) target gene in a miniaturized HDA-zeolite-heater (HZH) by an exothermic reaction. The amplicon detection on the chip was performed under room temperature conditions – either by successive hybridization and enzyme binding or by a combined step. A positive signal is displayed by enzymatically generated silver nanoparticle deposits, which serve as robust endpoint signals allowing an immediate visual readout. The hybridization assay enabled the reliable detection of 10 pg μL(-1) target DNA. This is the first report of an entirely electricity-free, field applicable detection approach for devastating Phytophthora species, exemplarily shown for P. kernoviae.

Background: The soil-borne pathogens Phytophthora nicotianae and P. capsici are the causal agents of root and stem rot of many plant species. Although P. capsici was considered the causal agent in one of the main pepper production areas of Spain to date, evidence of the presence of P. nicotianae was found. We aimed to survey the presence of P. nicotianae and study the variability in its populations in this area in order to improve the management of Tristeza disease., Results: A new specific primer and a TaqMan probe were designed based on the internal transcribed spacer regions of ribosomal DNA to detect and quantify P. nicotianae. Both morphological and molecular analysis showed its presence and confirmed it to be the causal agent of the Phytophthora disease symptoms in the studied area. The genetic characterization among P. nicotianae populations showed a low variability of genetic diversity among the isolates. Only isolates of the A2 mating type were detected., Conclusions: Not only is a specific and early detection of P. nicotianae essential but also the study of genetic variability among isolates for the appropriate management of the disease, above all, in producing areas with favorable conditions for the advance of the disease., (© 2014 Society of Chemical Industry.)

Background: Phytophthora blight caused by Phytophthora cajani is an emerging disease of pigeonpea (Cajanus cajan L.) affecting the crop irrespective of cropping system, cultivar grown and soil types. Current detection and identification methods for Phytophthora species rely primarily on cultural and morphological characteristics, the assessment of which is time-consuming and not always suitable. Sensitive and reliable methods for isolation, identification, zoospore production and estimating infection severity are therefore desirable in case of Phytophthora blight of pigeonpea., Results: In this study, protocols for isolation and identification of Phytophthora blight of pigeonpea were standardized. Also the method for zoospore production and in planta infection of P. cajani was developed. Quantification of fungal colonization by P. cajani using real-time PCR was further standardized. Phytophthora species infecting pigeonpea was identified based on mycological characters such as growth pattern, mycelium structure and sporangial morphology of the isolates and confirmed through molecular characterization (sequence deposited in GenBank). For Phytophthora disease development, zoospore suspension of 1 × 10(5) zoospores per ml was found optimum. Phytophthora specific real-time PCR assay was developed using specific primers based on internal transcribed spacer (ITS) 1 and 2. Use of real-time PCR allowed the quantitative estimation of fungal biomass in plant tissues. Detection sensitivities were within the range of 0.001 pg fungal DNA. A study to see the effect of elevated CO₂ on Phytophthora blight incidence was also conducted which indicated no significant difference in disease incidence, but incubation period delayed under elevated CO₂ as compared to ambient level., Conclusion: The zoospore infection method for Phytophthora blight of pigeonpea will facilitate the small and large scale inoculation experiments and thus devise a platform for rapid and reliable screening against Phytophthora blight disease of pigeonpea. qPCR allowed a reliable detection and quantification of P. cajani in samples with low pathogen densities. This can be useful in early warning systems prior to potential devastating outbreak of the disease.

Phytophthora is one of the most important and aggressive plant pathogenic genera in agriculture and forestry. Early detection and identification of its pathways of infection and spread are of high importance to minimize the threat they pose to natural ecosystems. eDNA was extracted from soil and water from forests and plantations in the north of Spain. Phytophthora-specific primers were adapted for use in high-throughput Sequencing (HTS). Primers were tested in a control reaction containing eight Phytophthora species and applied to water and soil eDNA samples from northern Spain. Different score coverage threshold values were tested for optimal Phytophthora species separation in a custom-curated database and in the control reaction. Clustering at 99% was the optimal criteria to separate most of the Phytophthora species. Multiple Molecular Operational Taxonomic Units (MOTUs) corresponding to 36 distinct Phytophthora species were amplified in the environmental samples. Pyrosequencing of amplicons from soil samples revealed low Phytophthora diversity (13 species) in comparison with the 35 species detected in water samples. Thirteen of the MOTUs detected in rivers and streams showed no close match to sequences in international sequence databases, revealing that eDNA pyrosequencing is a useful strategy to assess Phytophthora species diversity in natural ecosystems.

Several isothermal amplification techniques recently have been developed that are tolerant of inhibitors present in many plant extracts, which can reduce the need for obtaining purified DNA for running diagnostic assays. One such commercially available technique that has similarities with real-time polymerase chain reaction (PCR) for designing primers and a labeled probe is recombinase polymerase amplification (RPA). This technology was used to develop two simple and rapid approaches for detection of Phytophthora spp.: one genus-specific assay multiplexed with a plant internal control and the other species-specific assays for Phytophthora ramorum and P. kernoviae. All assays were tested for sensitivity (ranging from 3 ng to 1 fg of DNA) and specificity using DNA extracted from more than 136 Phytophthora taxa, 21 Pythium spp., 1 Phytopythium sp., and a wide range of plant species. The lower limit of linear detection using purified DNA was 200 to 300 fg of DNA in all pathogen RPA assays. Six different extraction buffers were tested for use during plant tissue maceration and the assays were validated in the field by collecting 222 symptomatic plant samples from over 50 different hosts. Only 56 samples were culture positive for Phytophthora spp. whereas 91 were positive using the Phytophthora genus-specific RPA test and a TaqMan real-time PCR assay. A technique for the generation of sequencing templates from positive RPA amplifications to confirm species identification was also developed. These RPA assays have added benefits over traditional technologies because they are rapid (results can be obtained in as little as 15 min), do not require DNA extraction or extensive training to complete, use less expensive portable equipment than PCR-based assays, and are significantly more specific than current immunologically based methods. This should provide a rapid, field-deployable capability for pathogen detection that will facilitate point-of-sample collection processing, thereby reducing the time necessary for accurate diagnostics and making management decisions.

We present methods for the detection and quantification of four Phytophthora species which are pathogenic on trees; Phytophthora ramorum, Phytophthora kernoviae, Phytophthora lateralis, and Phytophthora austrocedri. Nucleic acid extraction methods are presented for phloem tissue from trees, soil, and pure cultures on agar plates. Real-time PCR methods are presented and include primer and probe sets for each species, general advice on real-time PCR setup and data analysis. A method for sequence-based identification, useful for pure cultures, is also included.

Sudden oak death, the tree disease caused by Phytophthora ramorum, has significant environmental and economic impacts on natural forests on the U.S. west coast, plantations in the United Kingdom, and in the worldwide nursery trade. Stream baiting is vital for monitoring and early detection of the pathogen in high-risk areas and is performed routinely; however, little is known about the nature of water-borne P. ramorum populations. Two drainages in an infested California forest were monitored intensively using stream-baiting for 2 years between 2009 and 2011. Pathogen presence was determined both by isolation and polymerase chain reaction (PCR) from symptomatic bait leaves. Isolates were analyzed using simple sequence repeats to study population dynamics and genetic structure through time. Isolation was successful primarily only during spring conditions, while PCR extended the period of pathogen detection to most of the year. Water populations were extremely diverse, and changed between seasons and years. A few abundant genotypes dominated the water during conditions considered optimal for aerial populations, and matched those dominant in aerial populations. Temporal patterns of genotypic diversification and evenness were identical among aerial, soil, and water populations, indicating that all three substrates are part of the same epidemiological cycle, strongly influenced by rainfall and sporulation on leaves. However, there was structuring between substrates, likely arising due to reduced selection pressure in the water. Additionally, water populations showed wholesale mixing of genotypes without the evident spatial autocorrelation present in leaf and soil populations.

In the past decade several Phytophthora strains were isolated from diseased Pachysandra terminalis plants suffering stem base and root rot, originating from the Netherlands and Belgium. All isolates were homothallic and had a felt-like colony pattern, produced semi-papillate sporangia, globose oogonia and had a maximum growth at ~ 27 C. Several additional Phytophthora strains were isolated from diseased Buxus sempervirens plants, originating from the Netherlands and Belgium, which had sustained stem base and root rot; similar strains also were isolated from Acer palmatum, Choisya ternata and Taxus in the United Kingdom. All isolates were homothallic and had a stellate colony pattern, produced larger semi-papillate sporangia and smaller globose oogonia than the isolates from Pa. terminalis and had a maximum growth temperature of ~ 30 C. Phylogenetic analyses of both species using the internal transcribed spacer region of the nuc rDNA (ITS), mt cytochrome oxidases subunit I gene (CoxI) and nuc translation elongation factor 1-α gene (TEF1α) revealed that all sequences of each species were identical at each locus and unique to that species, forming two distinct clusters in subclade 2a. Sequence analysis of partial β-tubulin genes showed that both taxa share an identical sequence that is identical to that of Ph. himalsilva, a species originating from Asia, suggesting a common Asian origin. Pathogenicity trials demonstrated disease symptoms on their respective hosts, and re-isolation and re-identification of the inoculated pathogens confirmed Koch's postulates., (© 2015 by The Mycological Society of America.)

Phytophthora plurivora and other Phytophthora species are known to be serious pathogens of forest trees. Little is known, however, about the presence of P. plurivora in Polish oak forests and their role in oak decline. The aims of this study were to identify P. plurivora in healthy and declining Quercus robur stands in southern Poland and to demonstrate the relationship between different site factors and the occurrence of P. plurivora. In addition, the virulence of P. plurivora and other Phytophthora species was evaluated through inoculations using 2-year-old oak seedlings. Rhizosphere soil was investigated from 39 oak stands representing different healthy tree statuses. The morphology and DNA sequences of the internal transcribed spacer regions (ITS) of the ribosomal DNA and the mitochondrial cox1 gene were used for identifications. P. plurivora, an oak fine root pathogen, was isolated from rhizosphere soil samples in 6 out of 39 stands. Additionally, Phytophthora cambivora, Phytophthora polonica and Phytophthora rosacearum-like were also obtained from several stands. The results showed a significant association between the presence of P. plurivora and the health status of oak trees. Similar relationships were also observed for all identified Phytophthora species. In addition, there was evidence for a connection between the presence of all identified Phytophthora species and some site conditions. Phytophthora spp. occurred more frequently in declining stands and in silt loam and sandy loam soils with pH ≥ 3.66. P. plurivora and P. cambivora were the only species capable of killing whole plants, producing extensive necrosis on seedling stems.

Phytophthora citrophthora from citrus in eastern Corsica and Spain consists of distinct clonal lineages. In South Africa the extent of genetic variation among citrus-associated P. citrophthora isolates is unknown. This was investigated with isolates from South Africa (n =60), Spain (n =10) and six isolates representing three P. citrophthora groups CTR1, CTR2 and CTR3 previously identified with isozyme polymorphisms (Mchau and Coffey 1994). South African and Spanish isolates belonged to two lineages (G1, G2) based on an internal transcribed spacer (ITS) phylogeny, random amplified microsatellites (RAMS) and random amplified polymorphic DNA (RAPD) profiling. Although combined RAMS and RAPD data identified 14 genotypes, unweighted pair group method with arithmetic mean (UPGMA) analyses grouped the isolates into two clusters corresponding to lineages G1 and G2. Lineage G1 predominated among isolates from South Africa (92%) and Spain (100%). Phylogenetic analyses of the β-tubulin, cytochrome c oxidase subunit I (COX1) and ITS regions did not support the hypothesis that the two lineages represent distinct phylogenetic species but suggested that isozyme group CTR2 and possibly CTR3 are species distinct from P. citrophthora sensu stricto. Mating-type analyses, using tester strains from groups CTR2 and CTR3 revealed that most G1 lineage isolates (n =57) were sterile but that some were of the A1 mating type (n =8) whereas all G2 lineage isolates were A2 (n =5). The mating-type designation was confirmed with P. capsici tester strains. However, when A1 (G1 lineage) and A2 (G2 lineage including CTR1 reference isolates) mating-type isolates were paired in all possible combinations, no oogonia or antheridia were produced. This suggests that only tester strains P. capsici, CTR2 and CTR3 were able to produce sexual structures and that lineages G1 and G2 are sterile and reproductively isolated, which is supported by molecular data., (© 2014 by The Mycological Society of America.)

Nursery plants are important vectors for plant pathogens. Understanding what pathogens occur in nurseries in different production stages can be useful to the development of integrated systems approaches. Four horticultural nurseries in Oregon were sampled every 2 months for 4 years to determine the identity and community structure of Phytophthora spp. associated with different sources and stages in the nursery production cycle. Plants, potting media, used containers, water, greenhouse soil, and container yard substrates were systematically sampled from propagation to the field. From 674 Phytophthora isolates recovered, 28 different species or taxa were identified. The most commonly isolated species from plants were Phytophthora plurivora (33%), P. cinnamomi (26%), P. syringae (19%), and P. citrophthora (11%). From soil and gravel substrates, P. plurivora accounted for 25% of the isolates, with P. taxon Pgchlamydo, P. cryptogea, and P. cinnamomi accounting for 18, 17, and 15%, respectively. Five species (P. plurivora, P. syringae, P. taxon Pgchlamydo, P. gonapodyides, and P. cryptogea) were found in all nurseries. The greatest diversity of taxa occurred in irrigation water reservoirs (20 taxa), with the majority of isolates belonging to internal transcribed spacer clade 6, typically including aquatic opportunists. Nurseries differed in composition of Phytophthora communities across years, seasons, and source within the nursery. These findings suggest likely contamination hazards and target critical control points for management of Phytophthora disease using a systems approach.

Populations of the vegetable pathogen Phytophthora capsici are often highly diverse, with limited gene flow between fields. To investigate the structure of a newly established, experimental population, an uninfested research field was inoculated with two single-zoospore isolates of P. capsici in September 2008. From 2009 through 2012, ≈50 isolates of P. capsici were collected from the field each year and genotyped using five microsatellite loci. The same two isolates were also crossed in the lab. High levels of diversity were detected in the research field, with 26 to 37 unique multilocus genotypes detected each year. Through 2012, genotypic diversity did not decline and no evidence of genetic drift was observed. However, during the 2011 and 2012 growing seasons, four new alleles not present in either parental isolate were observed in the field. Selfing (but not apomixis) was observed at low frequency among in vitro progeny. In addition, evidence for loss of heterozygosity was observed in half of the in vitro progeny. These results suggest that recombination, mutation, and loss of heterozygosity can affect the genetic structure observed in P. capsici populations.

A molecular diagnostic assay for Phytophthora spp. that is specific, sensitive, has both genus- and species-specific detection capabilities multiplexed, and can be used to systematically develop markers for detection of a wide range of species would facilitate research and regulatory efforts. To address this need, a marker system was developed based on the high copy sequences of the mitochondrial DNA utilizing gene orders that were highly conserved in the genus Phytophthora but different in the related genus Pythium and plants to reduce the importance of highly controlled annealing temperatures for specificity. An amplification primer pair designed from conserved regions of the atp9 and nad9 genes produced an amplicon of ≈340 bp specific for the Phytophthora spp. tested. The TaqMan probe for the genus-specific Phytophthora test was designed from a conserved portion of the atp9 gene whereas variable intergenic spacer sequences were used for designing the species-specific TaqMan probes. Specific probes were developed for 13 species and the P. citricola species complex. In silico analysis suggests that species-specific probes could be developed for at least 70 additional described and provisional species; the use of locked nucleic acids in TaqMan probes should expand this list. A second locus spanning three tRNAs (trnM-trnP-trnM) was also evaluated for genus-specific detection capabilities. At 206 bp, it was not as useful for systematic development of a broad range of species-specific probes as the larger 340-bp amplicon. All markers were validated against a test panel that included 87 Phytophthora spp., 14 provisional Phytophthora spp., 29 Pythium spp., 1 Phytopythium sp., and 39 plant species. Species-specific probes were validated further against a range of geographically diverse isolates to ensure uniformity of detection at an intraspecific level, as well as with other species having high levels of sequence similarity to ensure specificity. Both diagnostic assays were also validated against 130 environmental samples from a range of hosts. The only limitation observed was that primers for the 340 bp atp9-nad9 locus did not amplify Phytophthora bisheria or P. frigida. The identification of species present in a sample can be determined without the need for culturing by sequencing the genus-specific amplicon and comparing that with a reference sequence database of known Phytophthora spp.

A non-papillate, heterothallic Phytophthora species first isolated in 2001 and subsequently from symptomatic roots, crowns and stems of 33 plant species in 25 unrelated botanical families from 13 countries is formally described here as a new species. Symptoms on various hosts included crown and stem rot, chlorosis, wilting, leaf blight, cankers and gumming. This species was isolated from Australia, Hungary, Israel, Italy, Japan, the Netherlands, Norway, South Africa, Spain, Taiwan, Turkey, the United Kingdom and United States in association with shrubs and herbaceous ornamentals grown mainly in greenhouses. The most prevalent hosts are English ivy (Hedera helix) and Cistus (Cistus salvifolius). The association of the species with acorn banksia (Banksia prionotes) plants in natural ecosystems in Australia, in affected vineyards (Vitis vinifera) in South Africa and almond (Prunus dulcis) trees in Spain and Turkey in addition to infection of shrubs and herbaceous ornamentals in a broad range of unrelated families are a sign of a wide ecological adaptation of the species and its potential threat to agricultural and natural ecosystems. The morphology of the persistent non-papillate ellipsoid sporangia, unique toruloid lobate hyphal swellings and amphigynous antheridia does not match any of the described species. Phylogenetic analysis based on sequences of the ITS rDNA, EF-1α, and β-tub supported that this organism is a hitherto unknown species. It is closely related to species in ITS clade 7b with the most closely related species being P. sojae. The name Phytophthora niederhauserii has been used in previous studies without the formal description of the holotype. This name is validated in this manuscript with the formal description of Phytophthora niederhauserii Z.G. Abad et J.A. Abad, sp. nov. The name is coined to honor Dr John S. Niederhauser, a notable plant pathologist and the 1990 World Food Prize laureate., (© 2014 by The Mycological Society of America.)

Phytophthora capsici from seven provinces of China were investigated for their mating type, hyphal growth, zoospore production, and virulence. All of the morphological characteristics and the results of polymerase chain reaction confirmed that these isolates were indeed Phytophthora capsici. The test of mating type showed that the mating types of 19 representative isolates from China varied. The hyphal growth and the amount of zoospores produced from these isolates differed and there was no evident relationship between them, which indicated the existence of genetic diversity among the isolates in China. Also, the isolates that were more virulent on the pepper cultivars that we checked produced more zoospores than other isolates.

Background: Phytophthora root and stem rot (PRR) of soybean, caused by Phytophthora sojae, is controlled by Rps genes. However, little is known regarding the Rps-induced molecular responses to P. sojae and how they actually overlap. We thus sequenced, analyzed, and compared the transcriptomes of 10 near isogenic lines (NILs), each with a unique Rps gene/allele, and the susceptible parent Williams, pre- and post-inoculation with the pathogen., Results: A total of 4,330 differentially expressed genes (DEGs) were identified in Williams versus 2,014 to 5,499 DEGs in individual NILs upon inoculation with the pathogen. Comparisons of the DEGs between the NILs and Williams identified incompatible interaction genes (IIGs) and compatible interaction genes (CIGs). Hierarchical cluster and heatmap analyses consistently grouped the NILs into three clusters: Cluster I (Rps1-a), Cluster II (Rps1-b, 1-c and 1-k) and Cluster III (Rps3-a, 3-b, 3-c, 4, 5, and 6), suggesting an overlap in Rps-induced defense signaling among certain NILs. Gene ontology (GO) analysis revealed associations between members of the WRKY family and incompatible reactions and between a number of phytohormone signaling pathways and incompatible/compatible interactions. These associations appear to be distinguished according to the NIL clusters., Conclusions: This study characterized genes and multiple branches of putative regulatory networks associated with resistance to P. sojae in ten soybean NILs, and depicted functional "fingerprints" of individual Rps-mediated resistance responses through comparative transcriptomic analysis. Of particular interest are dramatic variations of detected DEGs, putatively involved in ethylene (ET)-, jasmonic acid (JA)-, (reactive oxygen species) ROS-, and (MAP-kinase) MAPK- signaling, among these soybean NILs, implicating their important roles of these signaling in differentiating molecular defense responses. We hypothesize that different timing and robustness in defense signaling to the same pathogen may be largely responsible for such variations.

A previously unknown Phytophthora species was isolated from irrigation water in Virginia, USA. This novel species produces abundant noncaducous and nonpapillate sporangia in soil water extract solution. It sometimes produces chlamydospores and hyphal swellings in aged cultures and in Petri's solution. This species has optimum vegetative growth at 30 C and grows well at 35 C. The lowest and highest temperatures for growth are 5 and 40 C. All isolates examined in this study are compatibility type A1 and produce mostly plerotic oospores when paired with an A2 mating-type tester of P. cinnamomi. Sequence analyses of the rDNA internal transcribed spacer (ITS) regions and the mitochondrially encoded cytochrome c oxidase 1 (cox 1) gene placed this species in clade 9 of the genus Phytophthora. These characteristics support the description of this taxon as a new species for which we propose the name P. hydrogena sp. nov. Further phylogenetic and physiological investigations of clade 9 species revealed a high-temperature tolerant cluster including P. hydrogena, P. aquimorbida, P. hydropathica, P. irrigata, P. chrysanthemi, P. insolita, P. polonica and P. parsiana. These species all grow well at 35 C. The monophyly of the species in this heat-tolerant cluster except P. insolita and P. polonica is highly supported by the maximum-likelihood analyses of the ITS and cox 1 sequences.

Limited information is available on how soil and leaf populations of the sudden oak death pathogen, Phytophthora ramorum, may differ in their response to changing weather conditions, and their corresponding role in initiating the next disease cycle after unfavorable weather conditions. We sampled and cultured from 425 trees in six sites, three times at the end of a 3-year-long drought and twice during a wet year that followed. Soil was also sampled twice with similar frequency and design used for sampling leaves. Ten microsatellites were used for genetic analyses on cultures from successful isolations. Results demonstrated that incidence of leaf infection tripled at the onset of the first wet period in 3 years in spring 2010, while that of soil populations remained unchanged. Migration of genotypes among sites was low and spatially limited under dry periods but intensity and range of migration of genotypes significantly increased for leaf populations during wet periods. Only leaf genotypes persisted significantly between years, and genotypes present in different substrates distributed differently in soil and leaves. We conclude that epidemics start rapidly at the onset of favorable climatic conditions through highly transmissible leaf genotypes, and that soil populations are transient and may be less epidemiologically relevant than previously thought.

Phytophthora capsici causes significant loss to pepper production in China, and our objective was to investigate the population structure in Gansu province. Between 2007 and 2011, 279 isolates were collected from pepper at 24 locations. Isolates (or subsets) were assessed for simple sequence repeat (SSR) genotype, metalaxyl resistance, mating type, and physiological race using cultivars from the World Vegetable Center (AVRDC) and New Mexico recombinant inbred lines (NMRILs). The A1 and A2 mating types were recovered from nine locations and metalaxyl-resistant isolates from three locations. A total of 104 isolates tested on the AVRDC panel resolved five physiological races. None of 42 isolates tested on the NMRIL panel caused visible infection. SSR genotyping of 127 isolates revealed 59 unique genotypes, with 42 present as singletons and 17 having 2 to 13 isolates. Isolates with identical genotypes were recovered from multiple sites across multiple years and, in many cases, had different race types or metalaxyl sensitivities. Isolates clustered into three groups with each group having almost exclusively the A1 or A2 mating type. Overall it appears long-lived genetically diverse clonal lineages are dispersed across Gansu, outcrossing is rare, and functionally important variation exists within a clonal framework.

Pyrosequencing analysis was performed on soils from Italian chestnut groves to evaluate the diversity of the resident Phytophthora community. Sequences analysed with a custom database discriminated 15 pathogenic Phytophthoras including species common to chestnut soils, while a total of nine species were detected with baiting. The two sites studied differed in Phytophthora diversity and the presence of specific taxa responded to specific ecological traits of the sites. Furthermore, some species not previously recorded were represented by a discrete number of reads; among these species, Phytophthora ramorum was detected at both sites. Pyrosequencing was demonstrated to be a very sensitive technique to describe the Phytophthora community in soil and was able to detect species not easy to be isolated from soil with standard baiting techniques. In particular, pyrosequencing is an highly efficient tool for investigating the colonization of new environments by alien species, and for ecological and adaptive studies coupled with biological detection methods. This study represents the first application of pyrosequencing for describing Phytophthoras in environmental soil samples., (© 2013 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.)

Many factors must be considered in order to develop and implement treatment systems to improve the microbial quality of surface water and prevent the accidental introduction of plant and human pathogens into vegetable crops. The efficacy of chlorine gas (Cl2(g)) and chlorine dioxide (ClO2) injection systems in combination with rapid sand filtration (RSF) was evaluated in killing fecal indicator microorganisms in irrigation water in a vegetable-intensive production area. The efficacy of ClO2 and Cl2(g) was variable throughout the distribution systems and coliform bacteria never dropped below levels required by the United States Environmental Protection Agency for recreational waters. Sampling date and sampling point had a significant effect on the abundance of coliforms in Cl2(g)- and ClO2-treated water. Sampling date and sampling point also had a significant effect on the abundance of generic Escherichia coli in Cl2(g) treated water but only sampling point was significant in ClO2 treated water. Although the waterborne plant pathogen Phytophthora capsici was detected in five different sources of surface irrigation water using baiting and P. capsici-specific PCR, in vitro studies indicated that ClO2 at concentrations similar to those used to treat irrigation water did not reduce mycelial growth or direct germination of P. capsici sporangia and reduced zoospore populations by less than 50%. This study concludes that injection of ClO2 and Cl2(g) into surface water prior to rapid sand filtration is inadequate in reducing fecal indicator microorganism populations and ClO2 ineffectively kills infectious propagules of P. capsici. Additional research is needed to design a system that effectively targets and significantly reduces both plant and human pathogens that are present in surface irrigation water. A model for a multiple barrier approach to treating surface water for irrigation is proposed., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Phytophthora lateralis is a fungus-like (oomycete) pathogen of trees in the family Cupressaceae, including Chamaecyparis lawsoniana (Lawson cypress or Port Orford cedar). Known in North America since the 1920s, presumably having been accidentally introduced from its assumed East Asian centre of origin, until recently, this pathogen has not been identified causing disease in Europe except for a few isolated outbreaks. However, since 2010, there have been several reports of infection of C. lawsoniana by P. lateralis in the United Kingdom, including Northern Ireland. We sequenced the genomes of four isolates of P. lateralis from two sites in Northern Ireland in 2011. Comparison with the closely related tree and shrub pathogen P. ramorum (cause of ramorum disease of larch and other species in the UK) shows that P. lateralis shares 91.47% nucleotide sequence identity over the core conserved compartments of the genome. The genomes of the four Northern Ireland isolates are almost identical, but we identified several single-nucleotide polymorphisms (SNPs) that distinguish between isolates, thereby presenting potential molecular markers of use for tracking routes of spread and in epidemiological studies. Our data reveal very low rates of heterozygosity (compared with P. ramorum), consistent with inbreeding within this P. lateralis population., (© 2013 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.)

Surveys of Australian and South African rivers revealed numerous Phytophthora isolates residing in clade 6 of the genus, with internal transcribed spacer (ITS) gene regions that were either highly polymorphic or unsequenceable. These isolates were suspected to be hybrids. Three nuclear loci, the ITS region, two single copy loci (antisilencing factor (ASF) and G protein alpha subunit (GPA)), and one mitochondrial locus (cytochrome oxidase c subunit I (coxI)) were amplified and sequenced to test this hypothesis. Abundant recombination within the ITS region was observed. This, combined with phylogenetic comparisons of the other three loci, confirmed the presence of four different hybrid types involving the three described parent species Phytophthora amnicola, Phytophthora thermophila, and Phytophthora taxon PgChlamydo. In all cases, only a single coxI allele was detected, suggesting that hybrids arose from sexual recombination. All the hybrid isolates were sterile in culture and all their physiological traits tended to resemble those of the maternal parents. Nothing is known regarding their host range or pathogenicity. Nonetheless, as several isolates from Western Australia were obtained from the rhizosphere soil of dying plants, they should be regarded as potential threats to plant health. The frequent occurrence of the hybrids and their parent species in Australia strongly suggests an Australian origin and a subsequent introduction into South Africa., (Copyright © 2013 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.)

The most significant threat to pepper production worldwide is the Phytophthora blight, which is caused by the oomycete pathogen, Phytophthora capsici Leonian. In an effort to help control this disease, we isolated and characterized a P. capsici resistance gene, CaRGA2, from a high resistant pepper (C. annuum CM334) and analyzed its function by the method of real-time PCR and virus-induced gene silencing (VIGS). The CaRGA2 has a full-length cDNA of 3,018 bp with 2,874 bp open reading frame (ORF) and encodes a 957-aa protein. The protein has a predicted molecular weight of 108.6 kDa, and the isoelectric point is 8.106. Quantitative real-time PCR indicated that CaRGA2 expression was rapidly induced by P. capsici. The gene expression pattern was different between the resistant and susceptible cultivars. CaRGA2 was quickly expressed in the resistant cultivar, CM334, and reached to a peak at 24 h after inoculation with P. capsici, five-fold higher than that of susceptible cultivar. Our results suggest that CaRGA2 has a distinct pattern of expression and plays a critical role in P. capsici stress tolerance. When the CaRGA2 gene was silenced via VIGS, the resistance level was clearly suppressed, an observation that was supported by semi-quantitative RT-PCR and detached leave inoculation. VIGS analysis revealed their importance in the surveillance to P. capsici in pepper. Our results support the idea that the CaRGA2 gene may show their response in resistance against P. capsici. These analyses will aid in an effort towards breeding for broad and durable resistance in economically important pepper cultivars.

Studies were conducted to determine how Phytophthora cinnamomi survives during hot and dry Mediterranean summers in areas with limited surviving susceptible hosts. Two Western Australian herbaceous perennials Chamaescilla corymbosa and Stylidium diuroides and one Western Australian annual Trachymene pilosa were collected weekly from a naturally infested site from the Eucalyptus marginata (jarrah) forest from winter to spring and less frequently during summer 2011/2012. Selfed oospores, thick-walled chlamydospores, and stromata of P. cinnamomi were observed in each species. Oospores and thick-walled chlamydospores germinated in planta confirming their viability. This is the first report of autogamy by P. cinnamomi in naturally infected plants. Stromata, reported for the first time for P. cinnamomi, were densely aggregated inside host cells, and germinated in planta with multiple germ tubes with hyphae capable of producing oospores and chlamydospores. Trachymene pilosa was completely asymptomatic, S. diuroides did not develop root lesions but some plants wilted, whilst C. corymbosa remained asymptomatic above ground but lesions developed on some tubers. The presence of haustoria suggests that P. cinnamomi grows biotrophically in some hosts. Asymptomatic, biotrophic growth of P. cinnamomi in some annual and herbaceous perennials and the production of a range of survival structures have implications for pathogen persistence over summer and its management., (Copyright © 2013 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.)

Alder decline caused by Phytophthora alni has been one of the most important diseases of natural ecosystems in Europe during the last 20 years. The emergence of P. alni subsp. alni -the pathogen responsible for the epidemic-is linked to an interspecific hybridization event between two parental species: P. alni subsp. multiformis and P. alni subsp. uniformis. One of the parental species, P. alni subsp. uniformis, has been isolated in several European countries and, recently, in North America. The objective of this work was to assess the level of genetic diversity, the population genetic structure, and the putative reproduction mode and mating system of P. alni subsp. uniformis. Five new polymorphic microsatellite markers were used to contrast both geographical populations. The study comprised 71 isolates of P. alni subsp. uniformis collected from eight European countries and 10 locations in North America. Our results revealed strong differences between continental populations (Fst = 0.88; Rst = 0.74), with no evidence for gene flow. European isolates showed extremely low genetic diversity compared with the North American collection. Selfing appears to be the predominant mating system in both continental collections. The results suggest that the European P. alni subsp. uniformis population is most likely alien and derives from the introduction of a few individuals, whereas the North American population probably is an indigenous population.

Phytophthora nicotianae and P. cactorum cause Phytophthora rot of strawberry. A duplex real-time PCR technique for simultaneous detection and quantification of the two pathogens was developed. Species-specific primers for P. nicotianae and P. cactorum were designed based on the internal transcribed spacer regions (ITS) of rDNA and the ras-related protein gene Ypt1, respectively. TaqMan probes were labeled with FAM for P. nicotianae and HEX for P. cactorum. Specificities were demonstrated using 52 isolates, including various soil-borne pathogens. Sensitivities for P. nicotianae and P. cactorum DNAs were 10 fg and 1 pg, respectively. The technique was applied to naturally infested soil and root samples; the two pathogens were detected and the target DNA concentrations were quantified. Significant correlations of DNA quantities in roots and the surrounding soils were found. The minimum soil DNA concentration predicting the development of disease symptoms was estimated as 20 pg (g soil)(-1). In three strawberry greenhouses examined, the target DNA concentrations ranged from 1 to 1,655 pg (g soil)(-1) for P. nicotianae and from 13 to 233 pg (g soil)(-1) for P. cactorum. The method proved fast and reliable, and provides a useful tool to monitor P. nicotianae and P. cactorum in plants or soils.

Phytophthora root rot (PRR) of avocado (Persea americana), caused by Phytophthora cinnamomi, is the most serious disease of avocado worldwide. Previous studies have determined that this pathogen exhibits a primarily clonal reproductive mode but no population level studies have been conducted in the avocado-growing regions of California. Therefore, we used amplified fragment length polymorphism based on 22 polymorphic loci and mating type to investigate pathogen diversity from 138 isolates collected in 2009 to 2010 from 15 groves from the Northern and Southern avocado-growing regions. Additional isolates collected from avocado from 1966 to 2007 as well as isolates from other countries and hosts were also used for comparative purposes. Two distinct clades of A2 mating-type isolates from avocado were found based on neighbor joining analysis; one clade contained both newer and older collections from Northern and Southern California, whereas the other clade only contained isolates collected in 2009 and 2010 from Southern California. A third clade was also found that only contained A1 isolates from various hosts. Within the California population, a total of 16 genotypes were found with only one to four genotypes identified from any one location. The results indicate significant population structure in the California avocado P. cinnamomi population, low genotypic diversity consistent with asexual reproduction, potential evidence for the movement of clonal genotypes between the two growing regions, and a potential introduction of a new clonal lineage into Southern California.

Most Phytophthora spp. are destructive plant pathogens; therefore, effective monitoring and accurate early detection are important means of preventing potential epidemics and outbreaks of diseases. In the current study, a membrane-based oligonucleotide array was developed that can detect Phytophthora spp. reliably using three DNA regions; namely, the internal transcribed spacer (ITS), the 5' end of cytochrome c oxidase 1 gene (cox1), and the intergenic region between cytochrome c oxidase 2 gene (cox2) and cox1 (cox2-1 spacer). Each sequence data set contained ≈250 sequences representing 98 described and 15 undescribed species of Phytophthora. The array was validated with 143 pure cultures and 35 field samples. Together, nonrejected oligonucleotides from all three markers have the ability to reliably detect 82 described and 8 undescribed Phytophthora spp., including several quarantine or regulated pathogens such as Phytophthora ramorum. Our results showed that a DNA array containing signature oligonucleotides designed from multiple genomic regions provided robustness and redundancy for the detection and differentiation of closely related taxon groups. This array has the potential to be used as a routine diagnostic tool for Phytophthora spp. from complex environmental samples without the need for extensive growth of cultures.

Until recently Phytophthora lateralis was known only as the cause of dieback and mortality of Chamaecyparis lawsoniana in its native range in the Pacific Northwest (PNW). Since the 1990s however disease outbreaks have occurred increasingly on ornamental C. lawsoniana in Europe; and in 2007 the pathogen was discovered in soil around old growth Chamaecyparis obtusa in Taiwan, where it may be endemic. When the phenotypes of over 150 isolates of P. lateralis from Taiwan, across the PNW (British Columbia to California) and from France, the Netherlands and the UK were compared three growth rate groups were resolved: one slow growing from Taiwan, one fast growing from the PNW and Europe, and one of intermediate growth from a small area of the UK. Within these growth groups distinct subtypes were identified based on colony patterns and spore metrics and further discriminated in a multivariate analysis. The assumption that the three main growth groups represented phylogenetic units was tested by comparative sequencing of two mitochondrial and three nuclear genes. This assumption was confirmed. In addition two phenotype clusters within the Taiwan growth group were also shown to be phylogenetically distinct. These four phenotypically and genotypically unique populations are informally designated as the PNW lineage, the UK lineage, the Taiwan J lineage, and the Taiwan K lineage. Their characteristics and distribution are described and their evolution, taxonomic, and plant health significance is discussed., (Crown Copyright © 2012. Published by Elsevier Ltd. All rights reserved.)

Aims: To evaluate the accuracy of pyrosequencing for the description of Phytophthora communities in terms of taxa identification and risk of assignment for false Molecular Operational Taxonomic Units (MOTUs)., Methods and Results: Pyrosequencing of Internal Transcribed Spacer 1 (ITS1) amplicons was used to describe the structure of a DNA mixture comprising eight Phytophthora spp. and Pythium vexans. Pyrosequencing resulted in 16 965 reads, detecting all species in the template DNA mixture. Reducing the ITS1 sequence identity threshold resulted in a decrease in numbers of unmatched reads but a concomitant increase in the numbers of false MOTUs. The total error rate was 0·63% and comprised mainly mismatches (0·25%), Conclusions: Pyrosequencing of ITS1 region is an efficient and accurate technique for the detection and identification of Phytophthora spp. in environmental samples. However, the risk of allocating false MOTUs, even when demonstrated to be low, may require additional validation with alternative detection methods., Significance and Impact of the Study: Phytophthora spp. are considered among the most destructive groups of invasive plant pathogens, affecting thousands of cultivated and wild plants worldwide. Simultaneous early detection of Phytophthora complexes in environmental samples offers an unique opportunity for the interception of known and unknown species along pathways of introduction, along with the identification of these organisms in invaded environments., (© 2012 The Authors Letters in Applied Microbiology © 2012 The Society for Applied Microbiology.)

Two distinct subgroups (L2 and A(-2)) were recovered from irrigation reservoirs and a stream in Virginia, USA. After molecular, morphological and physiological examinations, the L2 subgroup was named Phytophthora aquimorbida and the A(-2) designated as Phytophthora taxon 'aquatilis'. Both taxa are homothallic. P. aquimorbida is characterized by its noncaducous and nonpapillate sporangia, catenulate and radiating hyphal swellings and thick-walled plerotic oospores formed in globose oogonia mostly in the absence of an antheridium. P. taxon 'aquatilis' produces plerotic oospores in globose oogonia mostly with a paragynous antheridium. It has semi-papillate, caducous sporangia with variable pedicels, but it does not have hyphal swelling. Analyses of ITS, CO1, β-tubulin and NADH1 sequences revealed that P. aquimorbida is closely related to P. hydropathica, P. irrigata and P. parsiana, and P. taxon 'aquatilis' is related to P. multivesiculata. The optimum temperature for culture growth is 30 and 20 C for P. aquimorbida and P. taxon 'aquatilis' respectively. Both taxa were pathogenic to rhododendron plants and caused root discoloration, pale leaves, wilting, tip necrosis and dieback. Their plant biosecurity risk also is discussed.

Background: The hydrolysis products of glucosinolates in Brassicaceae tissues are potentially useful for the control of fungal pathogens. The in vitro activity of these products against Phytophthora nicotianae Breda de Haan was studied, with the isolates exposed to the volatile products released from the brassica tissues., Results: The four species of Brassicaceae tested inhibited the mycelial growth of P. nicotianae isolates. The most effective developmental stage was different, depending on the species: buds yellowing in Sinapis alba L. and Brassica carinata A. Braun; seeds enlarging and all buds open in Brassica nigra (L.) W.D.J. Koch; cauliflower still covered by leaves in Brassica oleracea L. var. botrytis. At this stage, B. nigra and S. alba were the most effective (53.6 and 52.5% inhibition respectively). With all the biofumigants species tested, isolates from pepper plants were more susceptible to the brassica effect than those from tomato isolates., Conclusion: The developmental stage of plants has an influence on the biofumigant potential of Brassicaceae species against P. nicotianae. The isolates differ in susceptibility to compounds released, depending on their host of origin, suggesting the differentiation of populations of P. nicotianae in relation to the host of origin., (Copyright © 2012 Society of Chemical Industry.)

Phytophthora sojae is a devastating pathogen that causes soybean Phytophthora root rot. This study reports the development of a loop-mediated isothermal amplification (LAMP) assay targeting the A3aPro element for visual detection of P. sojae. The A3aPro-LAMP assay efficiently amplified the target element in < 80 min at 64 °C and was evaluated for specificity and sensitivity. The specificity was evaluated against P. sojae, Phytophthora spp., Pythium spp., and true fungi isolates. Magnesium pyrophosphate resulting from the LAMP of P. sojae could be detected by real-time measurement of turbidity. Phytophthora sojae DNA products were visualized as a ladder-like banding pattern on 2% gel electrophoresis. A positive colour (sky blue) was only observed in the presence of P. sojae with the addition of hydroxynaphthol blue prior to amplification, whereas none of other isolates showed a colour change. The detection limit of the A3aPro-specific LAMP assay for P. sojae was 10 pg μL(-1) of genomic DNA per reaction. The assay also detected P. sojae from diseased soybean tissues and residues. These results suggest that the A3aPro-LAMP assay reported here can be used for the visual detection of P. sojae in plants and production fields., (© 2012 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.)

Phytophthora borealis and Phytophthora riparia, identified in recent Phytophthora surveys of forest streams in Oregon, California and Alaska, are described as new species in Phytophthora ITS Clade 6. They are similar in growth form and morphology to P. gonapodyides and are predominantly sterile. They present unique DNA sequences, however, and differ in temperature/growth relations and geographic distribution.

The genus Phytophthora consists of many species that cause important diseases in ornamental, agronomic, and forest ecosystems worldwide. Molecular methods have been developed for detection and identification of one or several species of Phytophthora in single or multiplex reactions. In this article, we describe a padlock probe (PLP)-based multiplex method of detection and identification for many Phytophthora spp. simultaneously. A generic TaqMan polymerase chain reaction assay, which detects all known Phytophthora spp., is conducted first, followed by a species-specific PLP ligation. A 96-well-based microarray platform with colorimetric readout is used to detect and identify the different Phytophthora spp. PLPs are long oligonucleotides containing target complementary sequence regions at both their 5' and 3' ends which can be ligated on the target into a circular molecule. The ligation is point mutation specific; therefore, closely related sequences can be differentiated. This circular molecule can then be detected on a microarray. We developed 23 PLPs to economically important Phytophthora spp. based upon internal transcribed spacer-1 sequence differences between individual Phytophthora spp. Tests on genomic DNA of many Phytophthora isolates and DNA from environmental samples showed the specificity and utility of PLPs for Phytophthora diagnostics.

Phytophthora sojae is a destructive soilborne pathogen of soybean, but currently there is no rapid or commercially available testing for its infestation level in soil. For growers, such information would greatly improve their ability to make management decisions to minimize disease damage to soybean crops. Fatty acid profiling of P. sojae holds potential for determining the prevalence of this pathogen in soil. In this study, the Fatty Acid Methyl Ester (FAME) profile of P. sojae was determined in pure culture, and the profile was subsequently evaluated for its potential use in detecting the pathogen in soil. The predominant fatty acids in the FAME profile of P. sojae are the unsaturated 18C fatty acids (18:1ω9 and 18:2ω6) followed by the saturated and unsaturated 16C fatty acids (16:0 and 16:1ω7). FAME analysis of P. sojae zoospores showed two additional long-chain saturated fatty acids (20:0 and 22:0) that were not detected in the mycelium of this organism. Addition of a known number of zoospores of P. sojae to soil demonstrated that fatty acids such as 18:1ω9, 18:2ω6, 20:1ω9, 20:4ω6, and 22:1ω9 could be detected and quantified against the background levels of fatty acids present in soil. These results show the potential for using selected FAMEs of P. sojae as a marker for detecting this pathogen in soybean fields., (Copyright © 2012 British Mycological Society. Published by Elsevier Ltd. All rights reserved.)

Network theory has been applied to many aspects of biosciences, including epidemiology. Most epidemiological models in networks, however, have used the standard assumption of either susceptible or infected individuals. In some cases (e.g. the spread of Phytophthora ramorum in plant trade networks), a continuum in the infection status of nodes can better capture the reality of epidemics in networks. In this paper, a Susceptible-Infected-Susceptible model along a continuum in the infection status (SIS(c)) is presented, using as a case study directed networks and two parameters governing the epidemic process (probability of infection persistence (p(p)) and of infection transmission (p(t)). The previously empirically reported linear epidemic threshold in a plot of p(p) as a function of p(t) (Pautasso and Jeger, 2008) is derived analytically. Also the previously observed negative correlation between the epidemic threshold and the correlation between links in and out of nodes (Moslonka-Lefebvre et al., 2009) is justified analytically. A simple algorithm to calculate the threshold conditions is introduced. Additionally, a control strategy based on targeting market hierarchical categories such as producers, wholesalers and retailers is presented and applied to a realistic reconstruction of the UK horticultural trade network. Finally, various applications (e.g., seed exchange networks, food trade, spread of ideas) and potential refinements of the SIS(c) model are discussed., (Copyright © 2012 Elsevier Inc. All rights reserved.)

The effector gene Avr1b-1 of Phytophthora sojae determines the efficacy of the resistance gene Rps1b in soybean. The sequences of the Avr1b-1 locus in 34 Chinese isolates of P. sojae were obtained and analysed by polymerase chain reaction (PCR) and inverse PCR. Four different alleles and a complete deletion mutation of the Avr1b-1 gene were identified. Molecular analysis of the deletion breakpoints in the Avr1b-1 locus revealed that an 8-kb DNA sequence containing Avr1b-1 was deleted and a 12.7-kb DNA sequence was inserted at the same locus. A truncated transposase gene was found and five transposable elements were predicted in the inserted sequence, suggesting that the deletion of Avr1b-1 might be attributed to transposon movement. The transcription of Avr1b-1 was analysed in virulent isolates containing four alleles of Avr1b-1 by real-time reverse transcription-PCR. In all virulent isolates, only those isolates containing the second allele transcripted Avr1b-1., (© 2011 THE AUTHORS. MOLECULAR PLANT PATHOLOGY © 2011 BSPP AND BLACKWELL PUBLISHING LTD.)

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.

Little is known about indigenous Phytophthora species in natural ecosystems. Increasing evidence, however, suggests that a diverse, trophically complex Phytophthora community is important in many forests. The number of described species has steadily increased, with a dramatic spike in recent years as new species have been split from old and new species have been discovered through exploration of new habitats. Forest soil, streams, and the upper canopies of trees are now being explored for Phytophthora diversity, and a new appreciation for the ecological amplitude of the genus is emerging. Ten to twenty species are regularly identified in temperate forest surveys. Half or more of this Phytophthora diversity comes from species described since 2000. Taxa in internal transcribed spacer (ITS) Clade 6 are especially numerous in forest streams and may be saprophytic in this habitat. Three ecological assemblages of forest Phytophthora species are hypothesized: aquatic opportunists, foliar pathogens, and soilborne fine-root and canker pathogens. Aggressive invasive species are associated with all three groups.

Foliar hosts of Phytophthora ramorum are often susceptible to root infection but the epidemiological significance of such infections is unknown. A standardized test system was developed to quantify inoculum in runoff from root-infected Viburnum tinus ?Spring Bouquet? or Rhododendron ?Cunningham's White? cuttings. Cuttings of both species gave off a maximum amount of inoculum 1 to 3 weeks after inoculation. The greatest amount of inoculum was recovered from Viburnum roots that were 48 to 70 days old at the time of inoculation, or roots incubated at 15 to 20?C rather than 25?C. Inoculum in runoff from inoculated Viburnum roots was similar for four different isolates of P. ramorum representing both the NA1 and EU1 lineages. When Rhododendron cuttings were inoculated with P. ramorum, P. citricola, or P. cactorum, inoculum of all three pathogens was recovered from runoff, with the highest amount recovered from plants inoculated with P. citricola, followed by the other two. Compared with the other two pathogens, P. ramorum colonized root tissue to a smaller extent. The epidemiology of root infection by P. ramorum is important in itself but the assay might lend itself for use in risk analysis for root infection of other plant species and evaluation of control measures, and also shed light on other root-infecting Phytophthora spp.

The evolutionary history of Phytophthora cryptogea and P. drechsleri isolates previously collected from floriculture crops in North Carolina commercial greenhouses was explored with coalescent- and parsimony-based analyses. Initially, 68 isolates representing 13 location-host groups were sequenced at multiple loci. Sequences of all isolates within a group were identical. A subset of isolates were selected, cloned to resolve heterozygous sites, and analyzed with SNAP Workbench. The internal transcribed spacer (ITS) region of the ribosomal DNA and cytochrome oxidase II gene genealogies were congruent and indicated that P. cryptogea and P. drechsleri are sister species diverged from a common ancestor with no evidence of gene flow. In contrast, genealogies inferred from β-tubulin (β-tub) and translation elongation factor 1α (EF-1α) genes were in conflict with these loci. Coalescent analysis based on a nonrecombining partition in β-tub and EF-1α showed an initial (older) split between P. cryptogea and P. drechsleri, with a later (recent) event separating the remaining P. cryptogea haplotypes from P. drechsleri. A parsimony-based minimal ancestral recombination graph inferred recombination between P. cryptogea and P. drechsleri isolates in the ITS region and β-tub, suggesting genetic exchange between species. Also, putative recombination between A1 and A2 mating types of P. cryptogea suggests that sexual reproduction has occurred in the history of these P. cryptogea isolates.