Identification and Quantification of Pathogenic Pythium spp. from Soils in Eastern Washington Using Real-Time Polymerase Chain Reaction

ABSTRACT Traditional methods of quantifying Pythium spp. rely on the use of selective media and dilution plating. However, high variability is inherent in this type of enumeration and counts may not be representative of the pathogenic population of Pythium spp. Variable regions of the internal transcribed spacer of the rDNA were used to design species-specific primers for detection and quantification of nine Pythium spp. from soils in eastern Washington. Primer pairs were designed for Pythium abappressorium, P. attrantheridium, P. heterothallicum, P. irregulare group I, P. irregulare group IV, P. paroecandrum, P. rostratifingens, P. sylvaticum, and P. ultimum and used with real-time polymerase chain reaction. Standard curves were generated for each of the species using SYBR Green I fluorescent dye for detection of amplification. Seventy-seven isolates of Pythium were screened to confirm specificity of each primer set. DNA was extracted from soil and standard curves were generated for P. irregulare group I, P. irregulare group IV, and P. ultimum to correlate populations of each species in the soil with quantities of DNA amplified from the same soil. Examination of raw field soils revealed results similar to those observed in previous studies. This new technique for the quantification of Pythium spp. is rapid and accurate, and will be a useful tool in the future study of these pathogenic Pythium spp.     

Molecular characterization and pathogenicity of Pythium species associated with damping-off in greenhouse cucumber (Cucumis sativus) in Oman

A study was undertaken in 2004 and 2005 to characterize pathogens associated with damping-off of greenhouse-grown cucumber seedlings in 13 districts in Oman. Identification of Pythium to the species level was based on sequences of the internal transcribed spacer (ITS) of the ribosomal DNA. Of the 98 Pythium isolates collected during the survey, Pythium aphanidermatum , P. spinosum , P. splendens and P. oligandrum accounted for 76%, 22%, 1% and 1%, respectively. Pythium aphanidermatum was isolated from all of the districts, while P. spinosum was isolated from seven districts. Pathogenicity tests showed inter- and intraspecific variation in aggressiveness between Pythium species. Pythium aphanidermatum , P. spinosum and P. splendens were found to be highly aggressive at 25°C. However, the aggressiveness of P. spinosum decreased when the temperature was raised to 30°C, which was found to correspond to the lower frequency of isolation of P. spinosum in the warmer seasons, compared to the cooler time of the year. Pythium aphanidermatum exhibited limited intraspecific variation in the sequences of the ITS region of the rDNA and showed 100% similarity to the corresponding P. aphanidermatum sequences from GenBank. The ITS sequence data, as well as morphological characteristics of P. spinosum isolates, showed a high level of similarity within and between P. spinosum and P. kunmingense , and suggested that the two species were synonymous. This study represents the first report of P. spinosum, P. splendens and P. oligandrum in Oman.     

Molecular Diagnostics, Taxonomy, and Phylogeny of the Stem Nematode Ditylenchus dipsaci Species Complex Based on the Sequences of the Internal Transcribed Spacer-rDNA

ABSTRACT The stem nematode Ditylenchus dipsaci is of great economic importance worldwide as a parasite of agricultural crops and horticultural plants. The internal transcribed spacer (ITS) of rDNA from 23 populations of the D. dipsaci complex from various host plants were amplified and sequenced. Seven previously studied populations were also included in the study. The phylogenetic analysis of the full ITS and ITS2 sequence alignments using minimum evolution, maximum parsimony, and Bayesian inference under the complex model of DNA evolution revealed trees with two main clades: (i) D. dipsaci sensu stricto with diploid chromosome numbers and comprising most isolates from agricultural, ornamental, and several wild plants, and (ii) Ditylenchus spp. with polyploid chromosome numbers, reproductively isolated from diploid populations, and subdivided into six subclades ("giant race" from Vicia faba, Ditylenchus species parasitizing various Asteraceae, and a Ditylenchus sp. from Plantago maritima). Using the energy minimization approach and comparative sequence analysis, it has been found that the secondary structure of ditylenchid ITS2 is organized in three main domains. The importance of knowledge on the RNA structure for phylogenetic analysis is discussed. Conventional polymerase chain reaction (PCR) and real-time PCR with SYBR green dye I with a species specific primer have been developed for detection and quantification of D. dipsaci sensu stricto Validation tests revealed a rather high correlation between real numbers of fourth-stage juveniles of the stem nematodes in a sample and expected numbers detected by real-time PCR. Problems of accuracy of quantification are discussed.     

Identification of <Emphasis Type="Italic">Ditylenchus</Emphasis> species associated with Fabaceae seeds based on a specific polymerase chain reaction of ribosomal DNA-ITS regions

A technique based on the use of specific primers for polymerase chain reaction (PCR) was developed for the identification of the stem and bulb nematode belonging to the Ditylenchus dipsaci species complex. The internal transcribed spacer region ITS1 and ITS2, the gene 5.8 S and part of genes 18 S and 26 S of twenty populations of the D. dipsaci species complex belonging to both D. dipsaci sensu stricto and Ditylenchus sp. B (corresponding to populations of giant individuals associated to Vicia faba) and three congeneric species were amplified with two universal ribosomal primers. PCR-amplified DNA samples were digested with five restriction enzymes in order to reveal some polymorphism allowing the identification of D. dipsaci populations associated with Fabaceae seeds. The polymorphism among species was confirmed by the sequencing of the PCR products. A primer (DdpS2) was designed in a region conserved in all populations of both D. dipsaci sensu stricto and D. sp. B studied in the present work. The other Anguinidae species (except a few species from Central Asia associated to Astereaceae and D. sp. G associated to Plantago maritima) differ in two to four nucleotides at the 3′ extremity of this region. This sequence portion coincides with a TspEI restriction site. In combination with a primer located in the ribosomal region, this first primer is a good candidate for identification by PCR of populations of the D. dipsaci species complex found in Fabaceae seeds. A second primer (DdpS1) was designed in a similar way and was specific to D. dipsaci sensu stricto. The utility of these two sets of primers is discussed against the background of quarantine regulation.     

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.     

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.     

Pathogenicity of four Pythium species to wheat, barley, peas and lentils

Pythium heterothallicum, P. irregulare, P. torulosum and P. ultimum var. sporangiiferum were compared for pathogenicity to seedlings of winter wheat, spring barley, lentils and peas in growth chambers at 5, 10, 15, 20 and 25 C. These four fungi are among the most commonly isolated Pythium species from wheat roots and wheat-field soils in eastern Washington and northern Idaho, USA, where wheat, spring barley, lentils and peas are grown in various rotations. Pathogenicity was determined in artificially infested soils (500 propagules per g) based on ability to cause pre-emergence death and post-emergence stunting of seedlings. P. ultimum var. sporangiiferum caused significant pre-emergence death of the wheat at 15–25 C, lentils at 10–25 C. and peas at 5 25 C. P. irregulare caused pre-emergence death only of peas and only at 5 C. With the possible exception of lentils at 25 C, P. heterothallcum and P. torulosum caused no pre-emergence death of any of the four plant species. None of the species caused pre-emergence death of spring barley. P. ultimum var. sporangiiferum caused the most post-emergence stunting of wheat, peas and lentils at 10 C and above. Pythium irregulare caused as much or more stunting than P. ultimum var. sporangiiferum on wheat, lentils and peas at 5 C, and was the most pathogenic species on barley at 10, 20 and 25 C. P. irregulare caused significantly more post-emergence stunting of wheat at 5 C with than without chaff (added as a food base for the pathogen); this was not offset by adding ammonium sulphate with the chaff.     

Infection of Norway spruce (Picea abies (L.) Karst.) seedlings with Pythium irregulare Buism. and Pythium ultimum Trow.: histological and biochemical responses

We have studied the reaction of Picea abies seedlings to infection with Pythium. The highly virulent species Pythium ultimum and the less virulent species Pythium irregulare germinated on the root and hypocotyl surface, formed appressoria and penetrated through the stomata as well as through the epidermis. No major differences in the growth of both fungal species were observed during the early events of colonization. The less virulent species formed about 25% more appressoria suggesting that the fungus experienced difficulties with penetration. Differences were observed in the response of the host plant to infection. Autofluorescence, possibly related to deposition of lignin or lignin-like materials increased more in cortical and endodermal tissue colonized with the highly virulent P. ultimum than with the less virulent P. irregulare. Chitinase activity was highest in the tissues most extensively colonized by the fungus. In addition, a systemic increase of chitinase activity was also detected. Interestingly, chitinase activity increased systemically in cotyledons which were never in contact with the pathogen, indicating the translocation of a systemic signal. Salicylic acid was also detected in spruce seedlings; its level increased in roots during infection with the less virulent P. irregulare.     

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.     

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

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

Genetic variation among populations of Pythium irregulare in southern Australia

Isolates of Pythium irregulare were sampled from seven cereal crops throughout South Australia to determine the extent of genetic diversity within this pathogen and the scale of genetic differentiation among populations. Data derived from 29 individual restriction fragment length polymorphism (RFLP) loci differentiated 54 DNA fingerprints among the 92 isolates analysed. Some isolates had two alleles at several RFLP loci and were scored as heterozygous. One such isolate was selfed in vitro and segregation ratios in the progeny were not significantly different from those expected for allelic variation in a diploid. These data provided evidence that outcrossing occurs within P. irregulare and may contribute to the high level of genetic variation within the species ( D _T = 0·502). Allelic frequencies were significantly different among all seven populations and G _ST values showed significant genetic differentiation between populations. The average genetic identity among populations was low and hierarchical cluster analysis provided no clear evidence that populations formed geographically related groups. These analyses indicate low levels of interpopulation gene flow within P. irregulare and imply that population differentiation results from genetic drift.     

Molecular polymorphisms between populations of Pseudoperonospora cubensis from Greece and the Czech Republic and the phytopathological and phylogenetic implications

Molecular genetic polymorphisms within Pseudoperonospora cubensis isolates of different geographic origins were investigated to establish their phylogenetic relationships and to assess genetic variability between two distant pathogen populations. Thirty isolates originating from Greece (Crete; 15), the Czech Republic (13), the Netherlands (one) and France (one) were analysed by AFLP fingerprinting and ITS 5·8S rDNA sequence analysis. All isolates were obtained from cucumber ( Cucumis sativus ) plants showing typical downy mildew symptoms. Four AFLP primer combinations produced a total of 288 high-quality bands of which 45% were polymorphic, allowing isolates to be grouped into two separate clusters: one including the Central European (Czech Republic) and Western European (the Netherlands and France) and the other the Cretan isolates. Within each AFLP cluster there was some variation, which could be accounted for by geographic origin or pathogenicity. The two populations (Cretan vs. Central and Western European) exhibited a high degree of genetic isolation. There was no clear AFLP grouping of isolates on the basis of pathotypes. No variability was detected in the ITS1 region; however, ITS2 sequences grouped P. cubensis isolates in two subclusters: one with all investigated European and the other with Asian isolates. The two subclusters formed a larger P. cubensis cluster which was differentiated from the cluster of the neighbouring species Pseudoperonospora humuli . Within P. cubensis , AFLP fingerprints could resolve genetically isolated populations, even on small or medium geographic scales, while ITS2 sequence showed differences on a global scale, being only suitable for phylogenetic analyses.     

Composition and distribution of pythium communities in wheat fields in eastern washington state

ABSTRACT Pythium spp. were isolated from a mixture of soil and roots collected from 80 wheat fields in eastern Washington in the summer of 2000 from an area encompassing approximately 27,000 km(2). These sites covered a range of soil textures (coarse to fine, silty loess), average annual precipitation (200 to 600 mm), and average annual temperatures (7 to 11 degrees C). Soil type and annual precipitation run in an east-west gradient, while temperature has a north-south gradient. Species were identified using classical methods and by sequencing the internal transcribed spacer (ITS)-1 region of the rDNA and comparing these sequences to a database from a worldwide collection of Pythium spp. The species with the highest frequency of occurrence among all the sites were P. abappressorium sp. nov. (A) (50%), P. rostratum (R) (40%), P. debaryanum (D) (37.5%), P. heterothallicum (H) (33.7%), P.oligandrum (O) (31.2%), an unidentified P. sp. (aff. echinulatum) (E) (25%), and P. ultimum (U) (18%). P. intermedium, P. irregulare, P. paroecandrum, P. sylvaticum, P. dissimile, and P. dissoticum were isolated at a low frequency. From one to six species were isolated at each site, and there were 46 different species combinations detected. The species presence/absence data from all sites were analyzed with Jaccard's similarity coefficient hierarchical cluster analysis. Six communities were identified (species within each community designation in order of frequency among the sites within the community)-AD, AOU, AR, DEH, HE, and RU. In general, P. abappressorium was evenly distributed over all zones. AOU was more prevalent in zones with lower precipitation and coarser soil, while DEH and HE were associated with zones with higher precipitation and finer-textured soils on the basis of comparison of frequency distributions with the expected distribution over all the sites. The RU community was more prevalent in higher temperature zones. Canonical correspondence analysis was performed to examine the relationship between species and environmental variables. Soil type and precipitation were highly correlated with each other and with axis 1, which separated P. ultimum and P. abappressorium (lower variable values) from P. heterothallicum (higher variable values). Axis 2 and 3 were most correlated with temperature, and these axes separated P. oligandrum (higher value) from P. debaryanum (lower value) and P. ultimum-P. rostratum from the other species. The results suggest that Pythium species composition, distributions, and associations on a given crop may be influenced by environmental factors at a mesoscale level (100 to 1,000,000 ha).     

Comparative pathogenicity of Pythium species associated with poor seedling establishment of rice in Southern Australia

Isolates of Pythium attributable to thirteen named species and to species group F (sensu van der Plaats-Niterink) were isolated from roots of field-grown rice seedlings showing poor root development and lack of vigour. P. arrhenomanes and group F were the most commonly isolated species. The roots of 15-week-old rice plants yielded only P. flevoense, P. vanterpoolii, P. rostratum and Pythium group G of which the latter two were not obtained from seedling roots.
In pot tests with representative Pythium isolates, P. arrhenomanes was most pathogenic to rice seedlings, causing pre- and post-emergence death and average reductions in shoot and root growth in surviving seedlings of 48 and 70%, respectively. P. irregulare killed seedlings before but not after emergence, whilst the response of rice seedlings to P. myriotylum apparently depended on the length and timing of a cold shock treatment. P. pyrilobum did not reduce seedling number or shoot growth but reduced root growth. P. vanterpoolii and group F were not pathogenic in the tests. P. coloratum, P. echinulatum, P. flevoense, P. oedochilum, P. oligandrum, P. periilum, P. pyrilobum and P. rostratum were isolated infrequently and had no adverse effects on seedlings in pathogenicity tests, although P. myriotylum, P. oligandrum , and P. periilum were associated with significant increases in shoot growth. P. tracheiphilum was isolated from one site but its pathogenicity was not tested.
This is the first record of P. coloratum, P. echinulatum, P. flevoense, P. irregulare, P. oedochilum, P. oligandrum, P. periilum, P. pyrilobum and P. tracheiphilum from rice roots.     

Molecular Differentiation and Detection of Ginseng-Adapted Isolates of the Root Rot Fungus Cylindrocarpon destructans

ABSTRACT The soilborne fungus Cylindrocarpon destructans (teleomorph: Neonectria radicicola) causes root rot in a wide range of plant hosts; the disease is of particular concern in ginseng production, and in conifer and fruit tree nurseries. beta-Tubulin gene and rRNA gene internal transcribed spacer (ITS) sequence data and pathogenicity assays were used to characterize isolates of C. destructans from ginseng and other hosts. The results of these studies demonstrated a high amount of sequence divergence among strains identified as C. destructans or N. radicicola, suggesting the existence of several phylogenetic species in this complex. Accordingly, we propose that the two varieties of N. radicicola be raised to species status. Certain highly aggressive ginseng isolates from Ontario, Korea, and Japan have identical ITS and beta-tubulin sequences, and form a monophyletic clade (designated "clade a"); these strains are identified as C. destructans f. sp. panacis. Other ginseng strains clustered in monophyletic groups with strains from angiosperm and conifers. A subtractive hybridization method was used to isolate genomic DNA sequences with diagnostic potential from the aggressive C. destructans Ontario ginseng isolate 1640. One of these sequences was similar to the rRNA gene intergenic spacer from a Fusarium oxysporum isolate from Pinus ponderosa, and hybridized to DNA from F. oxysporum and all C. destructans isolates tested. Primers were designed that could be used to amplify this sequence specifically from the highly aggressive, ginsengadapted C. destructans isolates from Ontario and Korea and other members of clade a.     

Genetic analysis of eutypa strains from california supports the presence of two pathogenic species

ABSTRACT Eutypa dieback is a perennial canker disease that adversely affects grape (Vitis vinifera) production throughout the world. The causal agent has been known as either Eutypa armeniacae or E. lata, and it has been unclear whether the two taxa are separate species. We analyzed 115 isolates of Eutypa and conspecific strains, including 106 from California, using amplified fragment length polymorphism (AFLP) and sequence analysis of the ribosomal DNA (rDNA) internal transcribed spacer (ITS) sequence. Strains from cultivated plant species exhibited an average genetic distance of 0.34, as calculated by the DICE coefficient (NTSYS-pc software). An unweighted pair-group method with arithmetic averages dendrogram revealed a genetically distinct (distance of 0.73) group of Eutypa strains from valley oak (Quercus lobata) and madrone (Arbutus menziesii) and a strain from grape. Analysis of rDNA ITS sequences strongly supported the genetically distinct cluster detected in the AFLP data. Combined data indicated the presence of two species of Eutypa (E. armeniacae and E. lata) in our sample population. However, both Eutypa species were capable of infecting native and cultivated hosts, suggesting the potential for native tree species to serve as inoculum sources for grape infection in California. Further investigations of E. armeniacae and E. lata would contribute to the development of a successful disease management strategy.     

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.     

Evidence for the occurrence of natural hybridization in reed-associated Pythium species

A comparison of oomycete diversity in reed stands ( Phragmites australis ) of Lake Constance, Germany, and maize fields close by provided evidence for the occurrence of natural hybridization between Pythium phragmitis , a newly described reed pathogen, and an as-yet unknown Pythium species closely related to P. phragmitis and P. arrhenomanes . Internal transcribed spacer and β-tubulin sequences of a large set of Pythium isolates from reeds showed dimorphic signals at several positions, indicative of a mixture of characters of two parent species. Involvement of P. phragmitis in the putative hybrid species was confirmed after cloning and sequencing of ITS regions and β-tubulin genes of the hybrid isolates. Mitochondrially inherited cox II gene sequences did not show dimorphic sites and suggested that the hybridization event was relatively ancient, or that other species might be involved. Intermediate habitat preferences, morphological characters and aggressiveness towards reeds and other grasses confirmed the suggestion that these isolates comprise a natural hybrid between two Pythium species. Pythium arrhenomanes , likely to be involved in the putative hybrid's evolution, was repeatedly isolated from maize fields adjacent to P. phragmitis -infested reed stands. The interface between natural habitats with established oomycete communities and agricultural fields with potentially introduced pathogens might constitute an evolutionary hot-spot giving rise to new species with as-yet unknown host ranges. As indicated by inoculation tests, the hybrid was significantly more pathogenic towards reed rhizomes than P. phragmitis , which caused no damage to these organs. This is apparently the first report of the occurrence of natural hybridization in Pythium .     

What causes flag smut of wheat?

The causal agent of flag smut of wheat is currently subject to strict quarantine regulations in many countries and is believed to have a wide host range on wild and cultivated grasses. This fungus has been classified as both Urocystis agropyri and Urocystis tritici. Urocystis agropyri was first described from Elymus repens in Germany and U. tritici was first described from Triticum vulgare (=T. aestivum). In 1953, G. W. Fischer placed U. tritici and a large number of other Urocystis species in synonymy with U. agropyri. The present study is the first attempt to clarify the taxonomy and phylogeny of flag smut pathogens of grasses using molecular analyses. Three loci, the internal transcribed spacer (ITS) region of rDNA, the RNA polymerase II subunit 2 (RPB2), and translation elongation factor (TEF) protein‐coding regions were used for phylogenetic reconstruction to determine the species boundaries of 24 Urocystis specimens from triticoid hosts. Results indicate that there are several distinct lineages of flag smut pathogens, including the causal agent of flag smut of wheat, which is supported as a separate species, U. tritici. Sequences from specimens on E. repens, which are retained as U. agropyri, grouped in a clade distinct from those on wheat and rye. The closest relatives of U. tritici were found to be U. hispanica from Aegilops and Urocystis sp. from Thinopyrum junceiforme and Elymus trachycaulis. Recognition that U. tritici is genetically distinct from U. agropyri sensu stricto will impact regulatory policy and facilitate the development of diagnostic tests.     

Effects of root- and stolon-infecting fungi on root-colonizing nematodes of white clover

The effects on white clover ( Trifolium repens ) of different combinations of the nematodes Meloidogyne trifoliophila , Helicotylenchus dihystera and Heterodera trifolii and nine stolon-infecting and three root-infecting fungi were studied in a glasshouse experiment. The presence of the fungus Phytophthora megasperma alone increased ( P  < 0·001) root-rot severity and reduced ( P  < 0·001) plant growth. Other species combinations, such as Phoma nebulosa and Alternaria alternata , interacted and increased root-rot severity. Combinations of P. megasperma with Pythium irregulare , and P. nebulosa with Phoma medicaginis or A. alternata , increased M. trifoliophila populations. Several other fungi ( P. irregulare , P. nebulosa , Colletotrichum coccodes , Macrophomina phaseolina , P. medicaginis and Phoma sp.) interacted with the nematode M. trifoliophila causing severe root-knot symptoms. The results indicated that fungi and nematodes interacted to cause root and stolon rot and reduced yields, and that poor persistence of white clover in pastures is likely to be a problem with a complex etiology.