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.

Detection of Multiple Verticillium Species in Soil Using Density Flotation and Real-Time Polymerase Chain Reaction.

Wet sieving of soil samples, followed by plating on semi-selective medium and microscopic analysis, is the most commonly used technique to quantify microsclerotia-forming Verticillium species in soil. However, the method is restricted to small samples, does not allow easy differentiation between species, and takes several weeks to complete. This study describes an alternative method to test 100-g soil samples for three Verticillium species (V. tricorpus, V. dahliae, and V. longisporum) using density flotation-based extraction of microsclerotia followed by new real-time polymerase chain reaction (PCR) assays. Primers for these real-time PCR assays were designed to the ribosomal DNA internal transcribed spacer for V. tricorpus and the ß-tubulin gene for V. dahliae + V. longisporum and V. longisporum. Tests with artificially and naturally infested soils showed that the new method is reproducible and sensitive (0.1 to 0.5 microsclerotia/g soil), allows differentiation among the three species, and can be completed in one day. The results of the new method and the wet-sieving method were highly correlated for V. tricorpus (R2 = 0.78), but not for V. dahliae/V. longisporum, probably due to the loss of germinability of V. dahliae/V. longisporum microsclerotia during prolonged dry storage of the soil.

First Report of Phytophthora citricola on Cornus mas in Bulgaria.

Cornelian cherry dogwood (Cornus mas) is a widespread species in Bulgaria and some cultivars with large fruits are the subject of propagation. In the springs of 2007 and 2008, severe, unusual damages were observed on sporadically scattered plantlets of 'Kazanlashki' (known also as 'Kazanlaker') in a nursery located near Vratza in northwestern Bulgaria. Symptoms were identical in both years and expressed on the leaves, young shoots, and adjacent rootstock wood. Dark brown, necrotic leaf spots initiated most often from the leaf periphery and quickly covered more than half of the leaf area. Necrosis of the leaves and shoots spread toward the older woody tissues and the plantlets died within a couple of weeks. Isolations from symptomatic leaves, shoots, and rootstocks (three to five samples per plant organ) on potato dextrose agar always revealed a fungus-like organism that formed relatively fast-growing white, radial, petaloid colonies. Numerous, ovoid to obpyriform, noncaducous, semipapillate sporangia occasionally with two papilla were observed after 1 or 2 days of incubation at 20°C in nonsterile soil extract (1). Average sporangium size was 39 (35 to 45) × 31 (20 to 35) µm with a ratio between both parameters of approximately 1.26. The pathogen's paragynous antheridia and smooth-walled spherical oogonia (20 to 32 µm in diameter) yielded spherical aplerotic to almost plerotic oospores on V8 medium with an average size of 25 µm. The morphological data identified the organism as Phytophthora citricola (1). Isolates had identical cultural and morphological characteristics, and pathogenicity was tested by laboratory inoculations carried out in 2007 (two isolates) and twice in 2008 (three isolates). Separately, detached leaves of C. mas seedlings and 'Kazanlashki' were wiped with 70% ethanol, punctured with a needle, and the wounds inoculated with 5-mm mycelial plugs from a 7-day-old V8 growth plate. Sterile V8 plugs were placed onto similar wounds of control leaves. Leaf samples were incubated at 20°C in a humidified chamber. Necrosis similar to that observed in the field became visible around the mycelia plugs 4 days after inoculation. The necrotic lesions enlarged to 20 to 25 mm in diameter within the next 2 days, whereas the control leaves did not show any symptoms. Subsequently, the pathogen was reisolated solely from all the mycelium-inoculated samples. By means of the same inoculation method, pathogenicity was also demonstrated on shoots and mature fruits of C. mas. DNA was isolated from mycelium of an isolate and the internal transcribed spacer (ITS) region was amplified using ITS6 and ITS4 primers. The PCR product was sequenced (GenBank Accession No. FJ269034) and the BLAST search showed 100% homology with P. citricola, type II (2). To our knowledge, this is the first report of P. citricola on C. mas in Bulgaria, thus confirming its ability to attack Cornus spp. (3). Taking the lethal results of the disease and the polyphagous nature of the causal agent into consideration, this report is a serious warning for nurserymen and consumers. References: (1) D. C. Erwin and O. K. Ribeiro. Phytophthora Diseases Worldwide. The American Phytopathological Society, St. Paul, MN, 1996. (2) M. E. Gallegly and C. X. Hong. Phytophthora: Identifying Species by Morphology and DNA Fingerprints. The American Phytopathological Society, St. Paul, MN, 2008. (3) F. N. Martin and P. W. Tooley. Mycologia 95:269, 2003.

First Report of Phytophthora nicotianae on German Statice in Bulgaria.

In June of 2008, rapidly developing necrotic symptoms were observed on 2-month-old seedlings of German statice (Goniolimon tataricum, synonym Limonium tataricum) that were started from field-collected seeds and grown in plastic pots under greenhouse conditions in the region of Plovdiv, Bulgaria. Initial symptoms were slight yellowing and wilting of single, lower leaves. Subsequently, necrosis affected several petioles and stem bases, which led to complete plant collapse. Isolations from symptomatic petioles, stem bases, and main roots were performed on potato dextrose agar (PDA) and corn meal agar (CMA). When incubated at 24 to 25°C, white, round, arachnoid colonies with fluffy aerial mycelium developed on the PDA isolation plates, whereas the mycelium was less dense on CMA. Sporangia were formed sporadically when cultures were maintained on V8 medium and formed abundantly in nonsterile soil extract after 1 to 2 days of incubation at 20°C. Sporangia were noncaducous, ovoid to spherical, semipapillate (sometimes with two papilla), measured 47.5 to 65 µm (average 53.6 µm) × 35 to 53.5 µm (average 42.9 µm) with an average length/width ratio of 1.25:1. Terminal and intercalary chlamydospores (25 to 48 µm in diameter; average 37 µm) and hyphal swellings were also present. Maximum temperature for growth was 36°C. Pathogenicity of the presumable Phytophthora nicotianae (1) isolate was proved by placing 5-mm-diameter mycelial plugs of 7-day-old cultures grown on V8 medium onto the petiole bases of three 3-month-old G. tataricum plantlets. Each inoculation site was first wiped with 70% ethanol and then scalpel wounded. Sterile V8 plugs were used as controls and all inoculated sites were wrapped with Parafilm. The plantlets were incubated at room temperature (22 to 26°C) and the first necrotic lesions around the mycelial plugs appeared 5 to 7 days after inoculation. Plantlets collapsed approximately 2 weeks later. Additionally, three plantlets were inoculated under the same conditions by watering their stem bases with a 15-ml suspension of mycelium and spores obtained by washing 2-week-old V8 cultures with sterile distilled water. Within a 4-week period, the plantlets died due to a stem base and petiole necrosis. Simultaneously, the pathogen was reisolated from all inoculated samples but not from any control plants that were symptomless. The internal transcribed spacer (ITS) region of mycelial DNA was amplified (ITS6 and ITS4 primers) and the PCR product was sequenced (GenBank Accession No. FJ410333). BLAST analysis showed 100% homology with P. nicotianae. To our knowledge, this is the first report of P. nicotianae on G. tataricum in Bulgaria and one of the few reports from Europe of Phytophthora invasion of related Limonium species (2,3). References: (1) D. C. Erwin and O. K. Ribeiro. Phytophthora Diseases Worldwide. The American Phytopathological Society, St. Paul, MN, 1996. (2) E. Ilieva et al. Plant Dis. 85:445, 2001. (3) A. Pane et al. J. Plant Pathol. 87:301, 2005.

First Report of Phytophthora Stem Rot on Gloxinia in Bulgaria.

Severe stem base necrosis was observed on potted gloxinia (Sinningia speciosa) plants in a greenhouse in the Plovdiv Region of Bulgaria in the spring of 2006 and sporadically in 2007. Initial symptoms were water-soaked lesions at the stem base; eventually the lesions spread upward and downward until the entire stem was affected, resulting in withered leaves and plant collapse. Disease foci were sometimes apparent in rows of plants because of water splash. White, fungal-like colonies with arachnoid, aerial mycelia were obtained from affected plant tissue placed on potato dextrose agar (PDA) at 24 to 25°C in the dark. Pyriform to ellipsoid sporangia, 30 to 55 × 23 to 40 µm (average 38 × 27 µm), with a prominent papilla (sometimes two) and a short pedicel were observed. Chlamydospores were intercalary or terminal, spherical, and 15 to 55 µm in diameter. Oogonia and antheridia were not observed. On the basis of morphological features, the pathogen was tentatively identified as Phytophthora nicotianae (2). Pathogenicity was tested by placing 3-mm-diameter discs from 7-day-old PDA cultures onto wounded petioles of visibly healthy 3-month-old gloxinia potted plants (three replicates). Sterile PDA plugs were placed onto similar wounds of three control plants. The inoculated wounds were covered with Parafilm. Three days after inoculation, water-soaked lesions began to spread longitudinally in both directions on inoculated petioles. The pathogen was recovered from the inoculated tissue but not from the control plants. Molecular identification of the pathogen was achieved with PCR-restriction fragment length polymorphism of the internal transcribed spacer regions ITS1 and ITS2 of the ribosomal DNA (1). The restriction patterns obtained with the enzymes AluI, MspI, and TaqIα were identical to those of P. nicotianae, confirming the morphological identification. To our knowledge, this is the first report of P. nicotianae on gloxinia in Bulgaria. References: (1) D. E. L. Cooke and J. M. Duncan. Mycol. Res. 101:667, 1997. (2) D. C. Erwin and O. K. Ribeiro. Phytophthora Diseases Worldwide. The American Phytopathological Society, St. Paul, MN, 1996.

Disinfection of drain water of tomato by means of UV radiation and slow sand filtration in real greenhouse circumstances.

The efficiency of the disinfection of drain water was tested at 11 greenhouses with tomato cultivation on rockwool substrate in Flanders (Belgium) by means of mycological analysis. In addition the presence of phytopathogenic fungi in the drain water was analysed at 2 supplementary greenhouses with recirculation without disinfection.

The cytoskeleton in nematode feeding sites.

Sedentary edoparasitic nematodes induce specialised feeding cells in plant roots. Giant cells induced by root knot nematodes and syncytia generated by cyst nematodes in plant roots are large multinucleated cells containing a dense cytoplasm. To examine the plant cytoskeleton during feeding cell development, transcriptional activity of actin and tubulin genes and organization of the actin filaments and of the microtubules were analyzed in situ. Immunolocalizations of actins and tubulins and in vivo observation of green fluorescent protein decorated actin filaments and microtubules in nematode infected root cells revealed that major rearrangements of the cytoskeleton occur during the formation of nematode induced feeding cells.

Molecular characterisation of Arabidopsis lines tagged with a promoterless gus gene.

Promoter trapping has been performed through integration of a T-DNA containing a promoterless beta-glucuronidase (gus) reporter gene into the genome of Arabidopsis thaliana. A collection of T-DNA-tagged Arabidopsis lines has been produced with the vector pdeltagusBin19. Part of this collection was screened for lines with specific gus expression patterns. Here we report on the identification of two lines with gus expression in anthers and seeds. Both lines harbour complex T-DNA inserts. In one line, the integration of the T-DNA causes a male sterility phenotype and gus expression is developmentally regulated in anthers and flower bases. In the other line expression of gus is seen in the anther and seed endosperm.

Analysis of nematode-responsive promoters in sugar beet hairy roots.

One of the strategies to make crops resistant to the beet cyst nematode Heterodera schachtii is the destruction of the feeding site or syncytium. This can be achieved by local expression of the cytotoxic barnase gene under control of a nematode-inducible plant promoter that is active in the syncytium. Expression of barnase outside the feeding site has to be neutralized by its inhibitor barstar driven from a constitutive promoter that is downregulated in the syncytium. Several promoters that are upregulated in feeding structures were identified using the promoter tagging strategy in Arabidopsis thaliana (Barthels et al., 1997) or by differential cDNA screening in tomato (Van der Eycken et al., 1996). Nematode downregulated promoters in Arabidopsis were described by Goddijn et al. (1993). Five nematode-induced promoters (ARM1, 1164, 728, 25 and Lemmi9) and four downregulated promoters (CaMV35S, the nopaline synthase promoter (nos) and the rooting loci promoters RolC and RolD) fused to the beta-glucuronidase (gus) reporter gene were introduced into sugar beet hairy roots by transformation with Agrobacterium rhizogenes to evaluate their expression pattern. All upregulated promoters were found to be active at the base of lateral roots. The 728 and 25 promoter were as well active in root tips. In the 25-gus lines GUS could also be detected in the vascular tissue, while the ARM1 promoter was also active in sugar beet callus. The Lemmi9 promoter and the 4 constitutive promoters were active in the entire root. The transgenic hairy roots were inoculated with Heterodera schachtii and at different time-points (4, 8, 15, 22 days after inoculation; dpi) GUS analysis was performed on the infected roots. For the ARM1, 1164 and 728 promoter the highest gus expression level in syncytia was observed at 8 dpi. In 4 days old syncytia of the 25-gus lines the intensity of the GUS signal was of the same extent as the non-specific vascular signal. In later stages it even disappeared from the feeding sites. The gus expression level in syncytia of Lemmi9-gus hairy roots was equal to that in control roots. The RolC and 35S promoter were found to be downregulated at 8 dpi, the RolD promoter at 15 dpi and the nos promoter already at 4 dpi.

Comparison of human long-term bone marrow cultures (HLTBMCs) established from fresh and post-cryopreservation bone marrow samples from living and cadaver donors.

Human long-term bone marrow cultures (HLTBMCs) were established from thawed post-cryopreservation, as well as from cadaver donor bone marrow (BM) samples. The longevity was similar in the different series of HLTBMCs examined. CFU-GM could be cultured out of cadaver donor BM. This indicates that previously healthy people, under the conditions generally accepted as suitable for organ donation, could become suitable donors for allogeneic BM transplantation.

A quantitative and dynamic study of endothelial cells and megakaryocytes in human long-term bone marrow cultures.

The quantitative evolution of endothelial cells (ECs) in Dexter-type human long-term bone marrow cultures (HLTBMCs) was investigated. Using monoclonal antibodies directed against von Willebrand factor (vWF) and against membrane antigens (EN-4 and PAL-E), a low percentage--usually less than 1% of stromal cells--of ECs was detected in all confluent cultures established from 11 different bone marrow samples. Generally these cells are not associated directly with the areas of myelopoiesis ("cobblestone areas"). ECs cannot be demonstrated in the adherent layer of most young, non-confluent, and of some old, HLTBMCs. In some instances, morphological features suggestive of dynamic behavior were seen (sprouting, canal formation). In addition, a very low proportion of vWF-positive megakaryocytic cells was found in 4 of 11 cultures, always in direct contact with the stromal fibroblastic cells.