Béatrice Henricot

Improving ITS sequence data for identification of plant pathogenic fungi

SummaryPlant pathogenic fungi are a large and diverse assemblage of eukaryotes with substantial impacts on natural ecosystems and human endeavours. These taxa often have complex and poorly understood life cycles, lack observable, discriminatory morphological characters, and may not be amenable to in vitro culturing. As a result, species identification is frequently difficult. Molecular (DNA sequence) data have emerged as crucial information for the taxonomic identification of plant pathogenic fungi, with the nuclear ribosomal internal transcribed spacer (ITS) region being the most popular marker. However, international nucleotide sequence databases are accumulating numerous sequences of compromised or low-resolution taxonomic annotations and substandard technical quality, making their use in the molecular identification of plant pathogenic fungi problematic. Here we report on a concerted effort to identify high-quality reference sequences for various plant pathogenic fungi and to re-annotate incorrectly or insufficiently annotated public ITS sequences from these fungal lineages. A third objective was to enrich the sequences with geographical and ecological metadata. The results – a total of 31,954 changes – are incorporated in and made available through the UNITE database for molecular identification of fungi (http://unite.ut.ee), including standalone FASTA files of sequence data for local BLAST searches, use in the next-generation sequencing analysis platforms QIIME and mothur, and related applications. The present initiative is just a beginning to cover the wide spectrum of plant pathogenic fungi, and we invite all researchers with pertinent expertise to join the annotation effort.

Cylindrocladium buxicola, a new species affecting Buxus spp., and its phylogenetic status

A leaf and twig blight disease of Buxus spp. was found to be associated with a new species of Cylindrocladium. The novel species status was confirmed using morphological characters, sequencing of the ribosomal 5.8S RNA gene and the flanking internal transcribed spacers (ITS), the β-tubulin gene, and the high mobility group (HMG) of the MAT2 mating type gene. Cylindrocladium buxicola is proposed as a new name. Fifteen isolates from the UK and one isolate from New Zealand were paired in all combinations but no fertile perithecia were obtained suggesting that C. buxicola is heterothallic and all isolates belonged to one mating type. AFLP analysis showed that the isolates collected in the UK and New Zealand are genetically homogenous. Phylogenetic analyses indicated that this species falls within a new lineage.

Phytophthora niederhauserii sp. nov., a polyphagous species associated with ornamentals, fruit trees and native plants in 13 countries

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.

Phylogenetic analysis of the bluestain fungus Ophiostoma minus based on partial ITS rDNA and β-tubulin gene sequences

In an attempt to clarify the relationship between fungi classified as Ophiostoma minus, but of different geographic origins and mating systems, sequencing of the 5.8S and ITS 2 rDNA, and beta-tubulin gene was carried out. The beta-tubulin gene was highly informative, supporting the sub-division of O. minus into two groups based on geographic origin. Furthermore, isolates previously classified as O. pseudotsugae were confirmed as being clearly distinct from O. minus. However, sequencing did not reveal any polymorphisms between isolates with homothallic as compared to heterothallic mating systems. This was supported by crosses using methylbenzamidazole-2-yl carbamate nuclear markers which showed that hybridisation between isolates of different mating systems was possible. However, we propose that different mating systems may still signal a divergence of isolates of O. minus.

Phytophthora ramorum , the cause of sudden oak death or ramorum leaf blight and dieback

In 1993, a new species of Phytophthora was first recognised as the cause of a disease killing both nursery and mature rhododendrons in Germany and The Netherlands. The same fungus was observed attacking Viburnum in 1998 in Germany. It was named as P. ramorum in 2001 (Werres et al ., 2001). Since 1995, a disease of native coastal oaks ( Quercus and Lithocarpus spp.) has been observed in California and Oregon. A Phytophthora species was isolated from affected bark in 2000 and also confirmed as P. ramorum in 2001 (Rizzo et al ., 2002). The disease caused by P. ramorum was named in the American popular press ‘sudden oak death’ following the extensive mortality caused by this fungus on the native Californian oaks (Rizzo et al ., 2002). Since then the name of the disease has remained but with the increasing number of different hosts from which P. ramorum has been isolated and the range of symptoms, names such as ‘ramorum dieback’ and ‘ramorum leaf blight’ have been used to refer to the disease. The latin name ‘ramus’, meaning ‘branch’ refers to the pathogenicity of the fungus on branches (Werres et al ., 2001). The disease has been found in the following countries: Belgium, Canada, Denmark, France, Germany, Ireland, The Netherlands, Poland (interceptions), Norway, Slovenia, Spain, Sweden, UK, USA (California, Oregon and Washington). It is believed that the fungus has been introduced independently to northern Europe and western USA from an unidentified third country, i.e. it is not regarded as indigenous in either USA or Europe based on the recent discovery of the fungus, the differences in the population of the fungus between the two continents and the clonal structure of the sub-populations (Brasier, 2003; Rizzo & Garbelotto, 2003).

A reliable in vitro fruiting system for Armillaria mellea for evaluation of Agrobacterium tumefaciens transformation vectors

Armillaria mellea is a serious pathogen of horticultural and agricultural systems in Europe and North America. The lack of a reliable in vitro fruiting system for heterothallic A. mellea has hindered research and required dependence on intermittently available wild-collected basidiospores of endemic genotypes, necessitating the use of variable genetic material in transformation studies. Here we describe a reliable, reproducible in vitro fruiting method for heterothallic A. mellea from the western US. Isolates and growth conditions were evaluated to determine effective fruiting conditions. Following medium colonisation for 4 weeks, cultures were incubated under warm/bright conditions for 4-6 weeks before incubation in dim/cool conditions. Primordia emerged within 3-4 weeks following a temperature decrease and this was most efficient when coupled with a light reduction. Basidiocarps matured within 3-4 weeks and produced viable basidiospores. Agrobacterium tumefaciens and vectors were evaluated by transformation of in vitro-produced basidiospores and a versatile transformation vector was constructed to simplify promoter and marker gene exchange using homologous recombination in yeast. Fruiting bodies and viable basidiospores of A. mellea have been reliably produced in vitro which, coupled with the enhanced knowledge of suitable A. tumefaciens strains and vectors for transformation, will assist future genetic research into this important pathogen.

A native promoter and inclusion of an intron is necessary for efficient expression of GFP or mRFP in Armillaria mellea

Armillaria mellea is a significant pathogen that causes Armillaria root disease on numerous hosts in forests, gardens and agricultural environments worldwide. Using a yeast-adapted pCAMBIA0380 Agrobacterium vector, we have constructed a series of vectors for transformation of A. mellea, assembled using yeast-based recombination methods. These have been designed to allow easy exchange of promoters and inclusion of introns. The vectors were first tested by transformation into basidiomycete Clitopilus passeckerianus to ascertain vector functionality then used to transform A. mellea. We show that heterologous promoters from the basidiomycetes Agaricus bisporus and Phanerochaete chrysosporium that were used successfully to control the hygromycin resistance cassette were not able to support expression of mRFP or GFP in A. mellea. The endogenous A. mellea gpd promoter delivered efficient expression, and we show that inclusion of an intron was also required for transgene expression. GFP and mRFP expression was stable in mycelia and fluorescence was visible in transgenic fruiting bodies and GFP was detectable in planta. Use of these vectors has been successful in giving expression of the fluorescent proteins GFP and mRFP in A. mellea, providing an additional molecular tool for this pathogen.

A faster inoculation assay for Armillaria using herbaceous plants

ABSTRACT Armillaria (honey fungus) is a virulent necrotrophic pathogen that causes Armillaria root disease. Conventional Armillaria inoculation assays use young saplings as hosts and consequently are cumbersome, frequently conducted outdoors, and take many years from establishment to analysis of infection. We have developed and evaluated a faster inoculation assay for Armillaria that uses herbaceous plants as hosts, is carried out in controlled conditions, and reduces experimental durations to 3 months. Plant species of known susceptibility to Armillaria and comparisons between virulent A. mellea and opportunistic A. gallica were used to validate the assay. Mortality and diagnostic symptoms of Armillaria root disease such as epiphytic rhizomorphs and mycelial fans were used to assess levels of infection. We also attempted to reduce assay preparation time by substituting woody inocula with agar inocula, but typical symptoms of Armillaria root disease were only observed on plants infected with woody inocula. Through our assay, we identified five new potential herbaceous hosts of Armillaria: Kniphofia hirsuta, Hordeum vulgare, Lobelia cardinalis, Nicotiana tabacum and Helenium hoopesii – further expanding the extensive list of plants with susceptibility to Armillaria and suggesting infection of herbaceous species may be more widespread than currently acknowledged.

Morphological and Genetic Analyses of the Invasive Forest Pathogen Phytophthora austrocedri Reveal that Two Clonal Lineages Colonized Britain and Argentina from a Common Ancestral Population

Phytophthora austrocedri is causing widespread mortality of Austrocedrus chilensis in Argentina and Juniperus communis in Britain. The pathogen has also been isolated from J. horizontalis in Germany. Isolates from Britain, Argentina, and Germany are homothallic, with no clear differences in the dimensions of sporangia, oogonia, or oospores. Argentinian and German isolates grew faster than British isolates across a range of media and had a higher temperature tolerance, although most isolates, regardless of origin, grew best at 15°C and all isolates were killed at 25°C. Argentinian and British isolates caused lesions when inoculated onto both A. chilensis and J. communis; however, the Argentinian isolate caused longer lesions on A. chilensis than on J. communis and vice versa for the British isolate. Genetic analyses of nuclear and mitochondrial loci showed that all British isolates are identical. Argentinian isolates and the German isolate are also identical but differ from the British isolates. Single-nucleotide polymorphisms are shared between the British and Argentinian isolates. We concluded that British isolates and Argentinian isolates conform to two distinct clonal lineages of P. austrocedri founded from the same as-yet-unidentified source population. These lineages should be recognized and treated as separate risks by international plant health legislation.