Bioactive compounds produced by clones of Rhodiola rosea maintained in the Norwegian germplasm collection.

Roseroot, Rhodiola rosea, is a perennial herbaceous plant of the family Crassulaceae. The rhizomes of 95 roseroot clones in the Norwegian germplasm collection were analysed and quantified for their content of the bioactive compounds rosavin, salidroside, rosin, cinnamyl alcohol and tyrosol using HPLC analysis. All five bioactive compounds were detected in all 95 roseroot clones but in highly variable quantities. The ranges observed for the different compounds were for rosavin 2.90-85.95 mg g(-1), salidroside 0.03-12.85 mg g(-1), rosin 0.08-4.75 mg g(-1), tyrosol 0.04-2.15 mg g(-1) and cinnamyl alcohol 0.02-1.18 mg g(-1). The frequency distribution of the chemical content of each clone did not reflect a certain geographic region of origin or the gender of the plant. Significant correlations were found for the contents of several of these bioactive compounds in individual roseroot clones. A low, but not significant correlation was found between AFLP markers previously used to study the genetic diversity of the roseroot clones and their production of the chemical compounds. The maximum level of rosavin, rosin and salidroside observed were higher than for any roseroot plant previously reported in literature, and the roseroot clones characterized in this study might therefore prove to be of high pharmacological value.

Development of a highly sensitive nested-PCR method using a single closed tube for detection of Fusarium culmorum in cereal samples.

AIMS: The aim of the study was to develop a sensitive detection method of Fusarium culmorum contamination in cereal samples. METHODS AND RESULTS: A nested-PCR method using a single closed tube was developed for the detection of F. culmorum in infected cereal samples. The concentrations of the first primer pair was diluted 10,000 times compared to the concentration used for the second primer pair. Differing annealing temperatures allowed both first and second polymerase chain reaction (PCR) reactions to be performed subsequently in the same closed tube. The detection limit was 5-50 fg of purified target DNA and allowed the detection of 1% infected seeds of wheat in a mixture with uninfected grains. CONCLUSIONS: F. culmorum can be specifically detected in cereal samples by the highly sensitive method of nested-PCR in a single closed tube. SIGNIFICANCE AND IMPACT OF THE STUDY: This work describes the detection of F. culmorum in cereal samples that is approximately 100 times more sensitive than previous PCR methods, involves low risk of cross contaminations between samples, low costs and reduced hands-on time as compared to standard nested-PCR protocols.

Genetic variation, real-time PCR, metabolites and mycotoxins ofFusarium avenaceum and related species.

In this paper the latest studies dealing with genetic variation and mycotoxins ofF. avenaceum and related species are reviewed and compared to the data from chromatographic image analyses. Forty-three European strains ofFusarium avenaceum and related species were classified by chromatographic image analysis on full chromatographic matrices. The results were in most cases in agreement with those from morphological and molecular analyses and supported the separation betweenF. avenaceum, F. arthrosporioides andF. tricinctum and betweenF. avenaceum groups I and II. The mycotoxin profiles of the FinnishF. avenaceum, F. arthrosporioides andF tricinctum strains were very similar to each other. Moniliformin and enniatins were the main mycotoxins produced. A fluorogenic TaqMan PCR assay (qPCR) was used for the detection ofF. avenaceum/ F. arthrosporioides DNA in Finnish barley and wheat. The qPCR results obtained from grain samples were compared to mycotoxin levels. A correlation was found betweenF. avenaceum/F. arthrosporioides DNA and moniliformin (MON) and enniatin (ENNs) levels in barley. A correlation was also found between the combinedF. avenaceum/F. arthrosporioides/F. tricinctum contamination and MON and ENNs levels in barley in 2002, but not in 2003. This was probably due to the higher MON and ENNs levels in 2002 than in 2003. It was possible to use the DNA levels ofF. avenaceum/F. arthrosporioides to distinguish between most barley samples containing high amounts of MON and ENNs from those containing low levels of the mycotoxins.

First Report of Phytophthora ramorum in Ornamental Plants in Norway.

In November 2002, Phytophthora ramorum was isolated from Rhododendron catawbiense with wilted branches in a nursery in Bergen. The isolate was identified by characteristic deciduous, semipapillate sporangia, abundance of large chlamydospores, and slow growth (2). The identification was confirmed by ITS rDNA sequencing. After the first detection, the Norwegian Food Safety Authority (NFSA) started a survey of different ornamental plants during 2003. Of 21 samples from 10 locations, two rhododendron samples were positive. The rhododendron plants containing positive samples in 2002 and 2003 had been imported that same year as the disease was detected on them. In 2003, NFSA made regulations similar to those in the EU for P. ramorum, including the destruction of all infected plants and all plants susceptible to P. ramorum within a 2-m distance of the infected ones. The production of rhododendron in Norwegian nurseries is limited, and most rhododendrons marketed in the country are imported from March to May from other European countries. The main sale of rhododendron occurs in May and June, often before symptoms of P. ramorum are easy to observe. In 2004, 133 samples from 53 locations were analyzed. P. ramorum was found in 29 new locations. It was detected in 57 samples of rhododendron, in one sample of Pieris japonica, and one of Kalmia sp. Symptoms on pieris were similar to those on rhododendron with blighted twigs and leaf spots. In Kalmia sp., P. ramorum was isolated from small foliar spots. In 2005, special efforts were directed to detect P. ramorum before the spring sale. Between January and May, 142 samples were analyzed (including plants from 45 import shipments) and 19 yielded positive (including six samples from five import shipments). In 2005, 370 samples from 74 nurseries and garden centers were analyzed and 97 samples from 43 locations were positive (all were rhododendron). Ten of the 43 locations had been positive in 2004. Some of the samples that yielded positive in the summer and autumn came from import shipments or nurseries controlled earlier and found free from P. ramorum. As suggested previously, the disease is probably moving in trade as symptom-free plants (1) and also likely in batches with few infected plants with mild infections that are difficult to detect when random control is carried out in large shipments. Most nurseries receive new plants every year. It is thus difficult to determine if it is a reintroduction or an eradication failure when a nursery yields positive to P. ramorum in two consecutive years. In 2005, P. ramorum was detected on well-established Viburnum fragrans and rhododendron plants in a private garden in Bergen. The viburnum plants of this garden were heavily infected, with wilting of whole branches from the root collar to the top. The pathogen was also found on established rhododendron shrubs in four public greens in Bergen and two in Stavanger. The two cities are located at the southwestern coast of Norway and have more than 2,000 mm of annual precipitation, cool summers, and mild winters. The pathogenicity of 26 isolates from rhododendron, one from pieris, and one from Kalmia sp. was tested by placing mycelial plugs (18 isolates) or drops of zoospore suspension (7 isolates) on the unwounded abaxial surface of rhododendron leaves of cv. Cunninghams White. After 7 days, all isolates produced lesions larger then 2 cm at each inoculation site. P. ramorum was reisolated from the leaves. References: (1) C. M. Brassier et al. Mycol Res. 108:1107, 2004. (2) S. Werres et al. Mycol. Res. 105:1155, 2001.

Phylogenetic relationship of Fusarium langsethiae to Fusarium poae and Fusarium sporotrichioides as inferred by IGS, ITS, beta-tubulin sequences and UP-PCR hybridization analysis.

Fusarium langsethiae was recently described to accommodate "powdery" isolates of Fusarium poae, which morphologically resemble F. poae, but whose metabolite profile is similar to that of Fusarium sporotrichioides. In order to investigate the phylogenetic relationship of F. langsethiae to closely related species, we sequenced the internal transcribed spacer (ITS) regions 1 and 2 and part of the intergenic spacer (IGS) region of the rDNA cluster and part of the beta-tubulin gene from 109 strains of F. poae, F. sporotrichioides, F. langsethiae and Fusarium kyushuense from different geographic origin. Sequence analysis of ITS1 and 2 was unable to separate all F. sporotrichioides strains from F. langsethiae strains. Sequence analysis of beta-tubulin distinguished all four species, but it did not resolve the phylogenetic relationship between these two species. Sequence analysis of the IGS region distinguished the four species and led to a higher number of subgroups of the individual species, of which that of F. sporotrichioides var. minus isolates was even better supported than that of F. poae and F. langsethiae. Neighbor-joining and POY analyses of all combined sequences reliably separated all species studied, including F. langsethiae, clearly from F. sporotrichioides. The high intraspecific variability of the IGS sequences were found useful to group isolates according to their geographic origin. These results are in accordance with the results of the UP-PCR hybridization analysis. In summary, our data offer molecular support for the description of F. langsethiae as a new species in section Sporotrichiella.

An integrated taxonomic study of Fusarium langsethiae, Fusarium poae and Fusarium sporotrichioides based on the use of composite datasets.

An integrated systematic study was carried out to clarify the taxonomical position and relationship of Fusarium langsethiae to other taxa within the Fusarium section Sporotrichiella. Strains of this species were compared with strains of the closely related species Fusarium poae and Fusarium sporotrichioides using a composite dataset. This set consisted of DNA sequences derived from the ribosomal internal transcribed spacer (ITS) regions, partial sequences of the ribosomal intergenic spacer (IGS) region, the beta-tubulin and translation elongation factor-1 alpha (EF-1alpha) genes, AFLP fingerprints, chromatographic data on secondary metabolites and morphological data and growth characteristics. From these combined data, a consensus matrix was calculated by taking the mean of all pairwise distances between single isolates over all separate datasets. The consensus matrix was used as the basis for the construction of a UPGMA dendrogram and a multidimensional scaling, both of which revealed a clear separation of the three taxa. Partial IGS, EF-1alpha and beta-tubulin sequence-as well as chromatography-and AFLP-derived similarities turned out to be comparably consistent, while ITS sequence- and morphology-derived similarity matrices were rather divergent.

Isolation and characterization of a cDNA from Trichoderma harzianum P1 encoding a 14-3-3 protein homolog.

A full-length cDNA close, Th1433, (GenBank accession No. U24158), was isolated and characterized from the filamentous fungus, Trichoderma harzianum. The deduced amino acid (aa) sequence showed an acidic 30-kDa protein homologous to the 14-3-3 proteins, a family of putative kinase regulators originally characterized in mammalian brain tissue. The greatest homology, 71% identical aa, was found to BMH1, the corresponding protein from Saccharomyces cerevisiae and to the epsilon isoform from sheep brain. Southern analysis of genomic DNA indicated that Th1433 is a member of a small genomic family. At least two genes encoding 14-3-3-like proteins exist in T. harzianum. Northern analysis showed the highest level of expression during the first day after inoculation of the culture with conidial spores.

Primary structure of a novel barley gene differentially expressed in immature aleurone layers.

As a direct approach to elucidate the molecular biology of barley aleurone cell development, we differentially screened an aleurone cDNA library made from poly(A)+ RNA of immature grains for clones representing transcripts present in the aleurone but not in the starchy endosperm. For one of these clones, B22E, which hybridies to a 0.7 kb transcript, Northern and in situ hybridization revealed that expression is under complex spatial, temporal and hormonal control in barley grains. cDNAs corresponding to B22E transcripts were isolated from aleurone/pericarp and embryo of developing grains, and from germinating scutella. Among these were the nearly full-length aleurone/pericarp clone pB22E.a16 (541 bp). cDNAs matching the sequence of this clone (type 1 transcript) were found for all tissues investigated. In addition, cDNAs with an extra 12 bp insertion (type 2 transcript) were obtained from germinating scutella. The two different transcripts can encode novel barley proteins of 115 and 119 amino acids, respectively. A gene designated B22EL8 was isolated and sequenced; it encodes the type 1 B22E transcript and contains two introns of 145 and 125 bp. Particle bombardment of barley aleurone with a B22EL8 promoter-GUS (beta-glucuronidase) construct demonstrates that the promoter (3 kb) is active in developing barley grains. The promoter is not, however, active in the seeds of tobacco plants transgenic for the B22EL8 gene, indicating the existence of sequences specific for monocots. A comparison of 1.4 kb of upstream sequence of B22E with the maize c1 promoter reveals a number of short, identical sequences which may be responsible for aleurone cell-specific gene transcription.

Barley aleurone cell development: molecular cloning of aleurone-specific cDNAs from immature grains.

The cloning of 11 different homology groups of cDNAs representing genes expressed in aleurone, but not in starchy endosperm of 20-day-old barley grains is described. Among the cDNAs, four are aleurone-specific, while the remaining are also expressed in the embryo, but not in any other part of the plant.Sequence analysis of one of the aleurone-specific clones, B11E, reveals an open reading frame coding for an unidentified 10.4 kDa protein with a putative signal sequence and a possible metal-binding finger. The B11E gene has a high GC content in the 5' leader sequence (63%), as well as in the coding region (70%) compared to known cDNAs from the barley starchy endosperm. Northern analysis of B11E indicates maximum mRNA abundance around mid-phase of grain development.When isolated immature aleurone/pericarp is incubated in tissue culture medium (MS) the B11E message disappears, indicating a requirement for a diffusible factor from the intact grain for its continued presence.