P.J.M. Bonants

Phytophthora ramorum sp. nov., a new pathogen on Rhododendron and Viburnum

Since 1993, a hitherto unidentified Phytophthora species has been found associated with twig blight disease in Rhododendron and, sporadically, Viburnum. The morphology and growth characteristics of fourteen isolates from Germany and the Netherlands were investigated, together with their breeding system, the internal transcribed spacer (ITS) regions of the ribosomal DNA, amplified fragment length polymorphism (AFLP) fingerprints, and isozyme profiles, which were compared to those of a number of outgroup species. Morphologically the isolates are characterized by abundant production of chlamydospores and elongate, ellipsoid, deciduous sporangia with a short pedicel, in which they resemble P. palmivora. However, sporangia were semi-papillate, chlamydospores were much larger and cardinal temperatures much lower than those of P. palmivora. Oogonia with amphigynous antheridia and plerotic oospores were produced in dual cultures with an A2 mating type strain of P. cryptogea. ITS1 and ITS2 sequences of the unidentified species were closest to those of P. lateralis, but diered in three and eight nucleotides respectively from the latter species. AFLP fingerprints and isozyme patterns of malate dehydrogenase (MDH-2) and malic enzyme (MDHP) showed that the isolates formed a homogeneous group, distinct from all examined outgroup species, including P. lateralis. It was concluded that they represent a new Phytophthora species, described here as P. ramorum sp. nov. In pathogenicity tests all isolates of P. ramorum were pathogenic to Rhododendron.

Gene genealogies and AFLP analyses in the Fusarium oxysporum complex identify monophyletic and nonmonophyletic formae speciales causing wilt and rot disease

ABSTRACT The monophyletic origin of host-specific taxa in the plant-pathogenic Fusarium oxysporum complex was tested by constructing nuclear and mitochondrial gene genealogies and amplified fragment length polymorphism (AFLP)-based phylogenies for 89 strains representing the known genetic and pathogenic diversity in 8 formae speciales associated with wilt diseases and root and bulb rot. We included strains from clonal lineages of F. oxysporum f. spp. asparagi, dianthi, gladioli, lilii, lini, opuntiarum, spinaciae, and tulipae. Putatively nonpathogenic strains from carnation and lily were included and a reference strain from each of the three main clades identified previously in the F. oxysporum complex; sequences from related species were used as outgroups. DNA sequences from the nuclear translation elongation factor 1alpha and the mitochondrial small subunit (mtSSU) ribosomal RNA genes were combined for phylogenetic analysis. Strains in vegetative compatibility groups (VCGs) shared identical sequences and AFLP profiles, supporting the monophyly of the two single-VCG formae speciales, lilii and tulipae. Identical genotypes were also found for the three VCGs in F. oxysporum f. sp. spinaciae. In contrast, multiple evolutionary origins were apparent for F. oxysporum f. spp. asparagi, dianthi, gladioli, lini, and opuntiarum, although different VCGs within each of these formae speciales often clustered close together or shared identical EF-1alpha and mtSSU rDNA haplotypes. Kishino-Hasegawa analyses of constraints forcing the monophyly of these formae speciales supported the exclusive origin of F. oxysporum f. sp. opuntiarum but not the monophyly of F. oxysporum f. spp. asparagi, dianthi, gladioli, and lini. Most of the putatively nonpathogenic strains from carnation and lily, representing unique VCGs, were unrelated to F. oxysporum f. spp. dianthi and lilii, respectively. Putatively nonpathogenic or rot-inducing strains did not form exclusive groups within the molecular phylogeny. Parsimony analyses of AFLP fingerprint data supported the gene genealogy-based phylogram; however, AFLP-based phylogenies were considerably more homoplasious than the gene genealogies. The predictive value of the forma specialis naming system within the F. oxysporum complex is questioned.

DNA barcoding of oomycetes with cytochrome c oxidase subunit I and internal transcribed spacer

Oomycete species occupy many different environments and many ecological niches. The genera Phytophthora and Pythium for example, contain many plant pathogens which cause enormous damage to a wide range of plant species. Proper identification to the species level is a critical first step in any investigation of oomycetes, whether it is research driven or compelled by the need for rapid and accurate diagnostics during a pathogen outbreak. The use of DNA for oomycete species identification is well established, but DNA barcoding with cytochrome c oxidase subunit I (COI) is a relatively new approach that has yet to be assessed over a significant sample of oomycete genera. In this study we have sequenced COI, from 1205 isolates representing 23 genera. A comparison to internal transcribed spacer (ITS) sequences from the same isolates showed that COI identification is a practical option; complementary because it uses the mitochondrial genome instead of nuclear DNA. In some cases COI was more discriminative than ITS at the species level. This is in contrast to the large ribosomal subunit, which showed poor species resolution when sequenced from a subset of the isolates used in this study. The results described in this paper indicate that COI sequencing and the dataset generated are a valuable addition to the currently available oomycete taxonomy resources, and that both COI, the default DNA barcode supported by GenBank, and ITS, the de facto barcode accepted by the oomycete and mycology community, are acceptable and complementary DNA barcodes to be used for identification of oomycetes.

AFLP and phylogenetic analyses of North American and European populations of Phytophthora ramorum

The genetic structure within and between USA and European populations of the emerging phytopathogen Phytophthora ramorum was examined. Four primer combinations were used for amplified fragment length polymorphism (AFLP) fingerprinting of 67 USA isolates from California and Oregon, and 18 European isolates from Belgium, Germany, The Netherlands, Spain and the UK. In addition, three DNA regions (ITS, cox II, and nad 5) of additional Phytophthora species were amplified by polymerase chain reaction, sequenced, and analysed to provide better phylogenetic understanding of P. ramorum within the genus Phytophthora. AFLP banding patterns indicate that the 85 isolates form two distinct lineages within a monophyletic group, distinct from the closely related outgroup species P. lateralis. With the exception of two isolates from an Oregon nursery, European and USA isolates clustered separately within individual clades. The AFLP profiles also indicate that a single clonal lineage dominates the North American population, while the European population consists of an array of mainly unique, closely related AFLP types. Sequences from the three DNA regions were identical among all P. ramorum isolates, and phylogenetic analysis indicates that P. ramorum is closely related to P. lateralis and P. hibernalis.

Nonpathogenic Isolates of the Citrus Black Spot Fungus, Guignardia citricarpa, Identified as a Cosmopolitan Endophyte of Woody Plants, G. mangiferae (Phyllosticta capitalensis)

ABSTRACT The population structure of Guignardia citricarpa sensu lato (anamorph: Phyllosticta citricarpa), a fungus of which strains pathogenic to citrus are subject to phytosanitary legislation in the European Union and the United States, was investigated. Internal transcribed spacer sequences revealed two phylogenetically distinct groups in G. citricarpa. This distinction was supported by amplified fragment length polymorphism analysis that also supported the exclusion of two isolates that had apparently been misclassified as G. citricarpa. On cherry decoction agar, but not on other media, growth rates of group I isolates were lower than those of group II isolates. Conidial dimensions were similar, but group I isolates formed conidia with barely visible mucoid sheaths, whereas those of group II formed conidia with thick sheaths. Cultures of isolates belonging to group I produced rare infertile perithecia, whereas fertile perithecia were formed by most isolates of group II. Colonies of isolates belonging to group I were less dark than those of group II, with a wider translucent outer zone and a lobate rather than entire margin. On oatmeal agar, exclusively group I isolates formed a yellow pigment. Group I harbored strains from citrus fruits with classical black spot lesions (1 to 10 mm in diameter) usually containing pycnidia. Group II harbored endophytic strains from a wide range of host species, as well as strains from symptomless citrus fruits or fruits with minute spots (<2-mm diameter) without pycnidia. These observations support the historic distinction between slowly growing pathogenic isolates and morphologically similar fast-growing, nonpathogenic isolates of G. citricarpa. The latter proved to belong to G. mangiferae (P. capitalensis), a ubiquitous endophyte of woody plants with numerous probable synonyms including G. endophyllicola, G. psidii, P. anacardiacearum, and P. theacearum. G. mangiferae occurs in the European Union and the United States on many host species including citrus, and does not cause symptoms of citrus black spot, justifying its exclusion from quarantine measures.

Detection and identification of Phytophthora fragariae Hickman by the polymerase chain reaction

Phytophthora fragariae Hickman, which causes strawberry red stele and raspberry root rot, is a quarantine organism for which specific and sensitive detection methods are required to test the health of planting material. Sequences of the internal transcribed spacer regions of the ribosomal gene repeat (rDNA) were used to develop primers for P. fragariae in a nested Polymerase Chain Reaction (PCR). The fungus was readily detected in infected but symptomless roots by nested, but not single-round, PCR. It was also detected in infested water samples obtained from the Dutch General Inspection Service by nested PCR. Detection of PCR products was at least 10-fold more sensitive by PCR-ELISA than by conventional visualisation on agarose gels.

Diagnostic application of padlock probes—multiplex detection of plant pathogens using universal microarrays

Padlock probes (PLPs) are long oligonucleotides, whose ends are complementary to adjacent target sequences. Upon hybridization to the target, the two ends are brought into contact, allowing PLP circularization by ligation. PLPs provide extremely specific target recognition, which is followed by universal amplification and microarray detection. Since target recognition is separated from downstream processing, PLPs enable the development of flexible and extendable diagnostic systems, targeting diverse organisms. To adapt padlock technology for diagnostic purposes, we optimized PLP design to ensure high specificity and eliminating ligation on non-target sequences under real-world assay conditions. We designed and tested 11 PLPs to target various plant pathogens at the genus, species and subspecies levels, and developed a prototype PLP-based plant health chip. Excellent specificity was demonstrated toward the target organisms. Assay background was determined for each hybridization using a no-target reference sample, which provided reliable and sensitive identification of positive samples. A sensitivity of 5 pg genomic DNA and a dynamic range of detection of 100 were observed. The developed multiplex diagnostic system was validated using genomic DNAs of characterized isolates and artificial mixtures thereof. The demonstrated system is adaptable to a wide variety of applications ranging from pest management to environmental microbiology.

Natural Hybrids of Phytophthora nicotianae and Phytophthora cactorum Demonstrated by Isozyme Analysis and Random Amplified Polymorphic DNA

ABSTRACT Three similar isolates of Phytophthora (Phytophthora sp-h) were obtained from diseased Spathiphyllum and Primula plants. Cultural characteristics did not fit any known description of Phytophthora species. The Phytophthora sp-h isolates are papillate, are homothallic, possess 80 to 86% amphigynous antheridia, and have a maximum temperature for growth of 36.5 degrees C. Isozyme analysis of the Phytophthora sp-h isolates revealed a three-banded pattern with malic enzyme and a three-banded pattern with malate dehydrogenase on the second putative locus. The fastest band at both enzyme loci comigrated with the single P. nicotianae band, the slowest band comigrated with the single P. cactorum (and also P. pseudotsugae) band, and one band in between was concluded to represent the heterodimeric isozyme. The random amplified polymorphic DNA patterns of the Phytophthora sp-h isolates almost exclusively consisted of bands that were also present in either P. nicotianae or P. cactorum. Southern hybridization showed that bands specific for P. nicotianae were present as comigrating bands in the Phytophthora sp-h isolates. The same was found for species-specific bands of P. cactorum. It is concluded that the three Phytophthora sp-h isolates represent interspecific hybrids, P. nicotianae being the one parent and P. cactorum the other. Analysis of mito-chondrial DNA with restriction enzymes revealed banding patterns in all the Phytophthora sp-h isolates identical with those of P. nicotianae, confirming that indeed P. nicotianae was one of the parents.

Standardizing the Nomenclature for Clonal Lineages of the Sudden Oak Death Pathogen, Phytophthora ramorum

Phytophthora ramorum, the causal agent of sudden oak death and ramorum blight, is known to exist as three distinct clonal lineages which can only be distinguished by performing molecular marker-based analyses. However, in the recent literature there exists no consensus on naming of these lineages. Here we propose a system for naming clonal lineages of P. ramorum based on a consensus established by the P. ramorum research community. Clonal lineages are named with a two letter identifier for the continent on which they were first found (e.g., NA = North America; EU = Europe) followed by a number indicating order of appearance. Clonal lineages known to date are designated NA1 (mating type: A2; distribution: North America; environment: forest and nurseries), NA2 (A2; North America; nurseries), and EU1 (predominantly A1, rarely A2; Europe and North America; nurseries and gardens). It is expected that novel lineages or new variants within the existing three clonal lineages could in time emerge.

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.