Six new Phytophthora species from ITS Clade 7a including two sexually functional heterothallic hybrid species detected in natural ecosystems in Taiwan.

During a survey of Phytophthora diversity in natural ecosystems in Taiwan six new species were detected. Multigene phylogeny based on the nuclear ITS, ß-tubulin and HSP90 and the mitochondrial cox1 and NADH1 gene sequences demonstrated that they belong to ITS Clade 7a with P. europaea, P. uniformis, P. rubi and P. cambivora being their closest relatives. All six new species differed from each other and from related species by a unique combination of morphological characters, the breeding system, cardinal temperatures and growth rates. Four homothallic species, P. attenuata, P. flexuosa, P. formosa and P. intricata, were isolated from rhizosphere soil of healthy forests of Fagus hayatae, Quercus glandulifera, Q. tarokoensis, Castanopsis carlesii, Chamaecyparis formosensis and Araucaria cunninghamii. Two heterothallic species, P. xheterohybrida and P. xincrassata, were exclusively detected in three forest streams. All P. xincrassata isolates belonged to the A2 mating type while isolates of P. xheterohybrida represented both mating types with oospore abortion rates according to Mendelian ratios (4-33 %). Multiple heterozygous positions in their ITS, ß-tubulin and HSP90 gene sequences indicate that P. xheterohybrida, P. xincrassata and P. cambivora are interspecific hybrids. Consequently, P. cambivora is re-described as P. xcambivora without nomenclatural act. Pathogenicity trials on seedlings of Castanea sativa, Fagus sylvatica and Q. suber indicate that all six new species might pose a potential threat to European forests.

FUSARIUM HEAD BLIGHT SYMPTOM DISCRIMINATION: A USEFUL TOOL IN THE FIELD EVALUATION OF FUNGICIDE TREATMENTS.

Fusarium head blight (FHB) is one of the major diseases affecting wheat. It is caused by a complex of fungal species, resulting in yield losses and health problems due to mycotoxin production. The presence of multiple fungal species on wheat ears, with varying responses to active fungicide ingredients used in the field, makes the disease difficult to manage. In order to evaluate the efficacy of the timing of applications (at GS 39, GS 61 and GS 39+61) of a prothioconazole + fluoxastrobin-based fungicide, a 2-year field trial was conducted in Belgium. In both years, applications at GS 61 and GS 39+61 resulted in a significant reduction in symptom severity on ears and in deoxynivalenol (DON) content compared with the untreated control in 2013. In 2012, when Microdochium spp. were the most prevalent species, the treatment at GS 39 significantly reduced ear symptoms. Fusarium graminearum was predominant in the second year (2013) and caused significant DON accumulation in the grain after a single foliar spraying. The two genera were characterized by distinct types of symptoms: grouped bleached spikelet's for F. graminearum and isolated bleached spikelet's for Microdochium spp. This difference enabled the significant effect of the double treatments on symptoms caused by Microdochium spp. to be determined in the second year. This effect, which was also visible on leaf symptoms, suggests that Microdochium spp. epidemics in wheat might be polycyclic. Discrimination between symptoms caused by F. graminearum and Microdochium spp. could be a useful tool to study FHB management using fungicide treatments.

Biogeographical patterns and determinants of invasion by forest pathogens in Europe.

A large database of invasive forest pathogens (IFPs) was developed to investigate the patterns and determinants of invasion in Europe. Detailed taxonomic and biological information on the invasive species was combined with country-specific data on land use, climate, and the time since invasion to identify the determinants of invasiveness, and to differentiate the class of environments which share territorial and climate features associated with a susceptibility to invasion. IFPs increased exponentially in the last four decades. Until 1919, IFPs already present moved across Europe. Then, new IFPs were introduced mainly from North America, and recently from Asia. Hybrid pathogens also appeared. Countries with a wider range of environments, higher human impact or international trade hosted more IFPs. Rainfall influenced the diffusion rates. Environmental conditions of the new and original ranges and systematic and ecological attributes affected invasiveness. Further spread of established IFPs is expected in countries that have experienced commercial isolation in the recent past. Densely populated countries with high environmental diversity may be the weakest links in attempts to prevent new arrivals. Tight coordination of actions against new arrivals is needed. Eradication seems impossible, and prevention seems the only reliable measure, although this will be difficult in the face of global mobility.

First Report of the Ash Dieback Pathogen Hymenoscyphus pseudoalbidus (Anamorph Chalara fraxinea) on Fraxinus excelsior in Belgium.

Since the early 1990s, European ash (Fraxinus excelsior L.) has been affected by a lethal disease caused by the ascomycete fungus, Hymenoscyphus pseudoalbidus, originally known under the name of its anamorph, Chalara fraxinea (2,4). Pathogenicity of H. pseudoalbidus was demonstrated by inoculations on young trees (3). This emerging pathogen induces necrosis of leaf rachises, leaf wilting and shedding, bark necrosis, and wood discoloration as well as shoot, twig, and branch dieback. First observed in Poland, ash dieback now occurs in many parts of Europe. Since 2009, a survey of ash dieback caused by H. pseudoalbidus has been conducted in Wallonia (southern Belgium). Sampling units were selected to take the occurrence of ash stands and the potential points of entry of the pathogen into the country (nurseries, sawmills, rivers, and roads) into account. While the disease was not detected in 2009, young, naturally regenerated trees displaying typical symptoms of ash dieback were found in June 2010 in Silly, a village in the province of Hainaut. Symptomatic trees were located along a road in front of a large ash stand. Examination of shoots with bark necrosis from three symptomatic trees yielded positive results on the basis of a real time PCR test developed in our laboratory for the detection of H. pseudoalbidus (1). To confirm the molecular identification, fungal isolation from discolored wood onto malt extract agar supplemented with 100 mg liter-1 of streptomycin sulfate was attempted. After 18 days at 20 to 22°C in the dark, slow-growing, dull white colonies with gray patches, resembling those of C. fraxinea, had formed. The nuclear ribosomal internal transcribed spacer region (ITS) was amplified with primers ITS1 and ITS4 (4) and partly sequenced (GenBank Accession No. FR667687). A BLASTn search in GenBank revealed that the sequence of the Belgian isolate (452 bp) displayed 100% identity with sequences of a H. pseudoalbidus isolate from Switzerland (GenBank Accession No GU586932). In contrast, the sequence showed some mismatches with that of the closely related and probably strictly saprotrophic fungus, Hymenoscyphus albidus (GenBank Accession No GU586891.1). The strain was deposited as reference material in the Fungal Biology collection (CBS 128012). To our knowledge, this is the first report of ash dieback caused by H. pseudoalbidus in Belgium. The discovery of this aggressive tree pathogen in Wallonia documents its further westward spread in Europe. In the future, we expect that H. pseudoalbidus will continue its range expansion into areas that have so far not been affected by ash dieback. References: (1) A. Chandelier et al. For. Pathol. 40:87, 2010. (2) T. Kowalski. For. Pathol. 36:264, 2006. (3) T. Kowalski and O. Holdenrieder. For. Pathol. 39:1, 2009. (4) V. Queloz et al. For. Pathol. Online publication. doi:10.1111/j.1439-0329.2010.00645.x, 2010.

Involvement of Phytophthora species in the decline of beech Fagus sylvatica in Wallonia (Belgium).

During the last decade, typical symptoms of Phytophthora diseases were observed in beech stands of several European countries. The main symptoms were the presence of bleeding cankers on the stem, a low crown density as well as the yellowing of foliage and the small size of leaves. Several species of Phytophthora, such as Phytophthora citricola, P. cambivora and P. cactorum, were reported as the causal agents. In order to evaluate the implication of the different Phytophthora species in beech decline in the southern part of Belgium (Wallonia), a monitoring was undertaken with the help of managers of public and private forests. Phytophthora strains isolated from beech of different stands as well as from soil were characterized through morphological and molecular analyses (PCR-RFLP of ITS). All the isolated strains were identified as P. cambivora, except for one strain whose identification is ongoing. Molecular analysis was also directly applied to necrosed tissues of bleeding beeches and enabled the detection of additional cases. All positive cases exhibited a profile characteristic of the species P. cambivora, except for one of the sampled trees showing a different Phytophthora profile also corresponding to the unidentified isolated strain. Identification of the Phytophthora species linked to this different RFLP profile is also ongoing. Both complementation types (A1 and A2) of P. cambivora were identified, sometimes in the same sampling site. Ornamented oogonia characteristic of this species were produced by pairing A1 and A2 strains isolated from the same site.

Effect of temperature and bacteria on sporulation of Phytophthora alni in river water.

In order to evaluate the role of river water on the spread of the alder disease caused by Phytophthora alni, water samples were collected at different periods of the year 2004 in two rivers displaying contrasting biological quality indices. Sporangia were produced from isolates of P. alni belonging to the three subspecies, at the river temperature (between 8 and 15 degrees C according to the sampling period). The sporulation efficiency was evaluated according to a scale of 0-9, based on the amount of sporangia produced on mycelium plugs immersed in the water for two days. Sporulation was also evaluated in river water sterilised by filtration. The amount of sporangia increased with the water temperature for both rivers, regardless of the biological quality. No sporangium was produced at the lowest temperature (8 degrees C). Sterilisation of the water drastically reduced the sporangia-stimulating effect for most of the P. alni isolates.

First Report of Phytophthora ramorum on Viburnum bodnantense in Belgium.

In the past decade, a new Phytophthora species inducing shoot canker on Rhododendron and dieback of Viburnum has been observed in Europe, mainly in Germany and the Netherlands, and California. This new pathogen has been named Phytophthora ramorum (3). In May 2002, a diseased Viburnum plant (Viburnum bodnantense) from the Plant Protection Service (Ministry of Agriculture, Belgium) was submitted to our laboratory for diagnosis. Symptoms included wilting, leaves turning from green to brown, discolored vascular tissues, and root necrosis. The plant came from a Belgian ornamental nursery that obtained supplies of stock plants from the Netherlands. Pieces of necrotic root tissue were excised, surface-disinfected, and transferred aseptically to a Phytophthora selective medium. P. ramorum was identified based on morphological characteristics, including the production of numerous, thin-walled chlamydospores (25 to 70 µm in diameter, average 43 µm) and deciduous, semi-papillate sporangia arranged in clusters. Radial growth after 6 days at 20°C on V8 juice agar was 2.8 mm per day. Random amplified microsatellite markers (RAMS) (2) from the total genomic DNA of the Belgian strain (CBS 110901) were similar to those of P. ramorum reference strains (CBS 101330, CBS 101332, and CBS 101554). Using PCR primers specific for P. ramorum, the identification was confirmed by W. A. Man in't Veld (Plantenziektenkundige Dienst, Wageningen, the Netherlands) (1). A pathogenicity test was carried out on three sterile cuttings of Rhododendron catawbiense (3). Brown lesions were observed on the inoculated cuttings after 6 to 7 days. None of the three uninoculated cuttings showed symptoms of infection. P. ramorum was reisolated from lesion margins on the inoculated cuttings. To our knowledge, this is the first report of the fungus from Belgium. Since our initial observation, we have found P. ramorum in other Belgian nurseries on R. yakusimanum. References: (1) M. Garbelotto et al. US For. Ser. Gen. Tech. Rep. PSW-GRT. 184:765, 2002. (2) J. Hantula et al. Mycol. Res. 101:565, 1997. (3) S. Werres et al. Mycol. Res. 105:1155, 2001.

RT-PCR-ELOSA tests on pooled sample units for the detection of virus Y in potato tubers.

A highly sensitive RT-PCR protocol able to detect potato virus Y (PVY) in pooled sample units (tubers) was developed. PVY-specific primers selected in the coat protein gene were found to amplify a 359 bp fragment from diluted crude extract of infected tubers. For the detection of the amplification products, a colorimetric detection procedure in microtiter plates was established. The amplicons are hybridized between a covalently linked capture probe and a specific biotinylated detection probe ELOSA tests. This detection method detects at least 50 pg of virus per reaction for the four cultivars tested. The RT-PCR-ELOSA assay was adapted to pooled units in order to increase the sample size while reducing the number of tests.