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The prevalence of Rhizoctonia spp. in European soils was determined by analysing soil samples from 282 locations. Rhizoctonia spp. were found in 68% of these samples from France, Germany, the UK, Poland, Italy, Spain, Hungary and the Czech Republic. Samples from 136 locations were further analysed by pyrosequencing. Seventy-six percent of the isolates were Rhizoctonia solani and 24% binucleate Rhizoctonia spp. Rhizoctonia solani anastomosis group (AG) 5 was detected most frequently (25%), followed by AG 9 (16%) and AG 4 (13%). For the binucleate Rhizoctonia spp., AG E was most prevalent (13%). Rhizoctonia cerealis was not detected in soil samples. Soil type or cropping history had no effect on the type of Rhizoctonia observed. Rhizoctonia solani AG 5 was the most frequently detected AG irrespective of the previous crop. The spectrum of AGs detected was similar for France, Germany and Poland but was significantly different for the UK (P = 0·0016). Finally, the baseline sensitivity towards sedaxane, a new active ingredient for seed treatment, was analysed for all isolates. The results indicate a low baseline sensitivity (average EC50 of 0·028 p.p.m.) for all Rhizoctonia AGs. No difference in sensitivity was observed with the isolates obtained from different countries.

Polymorphisms between the coding sequences of high-molecular-weight (HMW) glutenin x-type genes at the Glu-1 locus were used to amplify Glu-1B x-type-specific PCR fragments. PCR analysis in a wheat cultivar subset carrying different Glu-1B x-type alleles resulted in PCR fragments that differed in size for Glu-B1-1d (B-x6) and non -Glu-B1-1d (B-x6) genotypes. Subsequent sequencing analysis revealed a 15-bp in-frame insertion in the coding regions of all Glu-B1-1d (B-x6) genotypes which allowed the development of a B-x6-specific PCR assay for high-throughput allele sizing by ion-pair reversed-phase high-performance liquid chromatography. The assay was validated in a set of 86 German wheat cultivars, and genotyping data unequivocally verified the presence of HMW glutenin subunits GLU-B1-1D (Bx-6) + GLU-B1-2A (By-8) by means of sodium dodecyl sulphate-polyacrylamide gel electrophoresis. These results demonstrate that the PCR assay can be applied for the detection and negative selection of the 'poor breadmaking quality' Glu-B1-1d (B-x6) alleles in wheat breeding programs.

Net blotch, caused by Pyrenophora teres, is a common disease of barley ( Hordeum vulgareL.). Two PCR-based differential screening techniques, cDNA-amplified fragment length polymorphism (cDNA-AFLP) and suppression subtractive hybridisation (SSH), were employed to clone cDNA copies of transcripts that are up-regulated during conidial germination. The nucleotide sequences of 35 transcripts were analysed, and the amino acid sequences of their predicted products were compared with entries in databases. Eleven of these clones showed homology to genes from other ascomycetes coding for a transcription factor, two regulatory proteins, a putative transposase, a protein required for the biogenesis of cytochrome C oxidase, a threonine synthase, a probable subunit of a phenylalanine-tRNA synthetase, a subunit of RNA polymerase I, a cation transport protein, a vacuolar ATP synthase subunit, and an RNA processing protein. One conserved hypothetical protein was found and 23 sequences could not be functionally classified. The relative expression of five transcripts at 0, 1, 2, 3, 6, 12 and 24 h after induction of germination was determined by real-time RT-PCR using 18S rRNA as the endogenous reference sequence. All transcripts showed a significant increase in expression during early stages of germination. The maximum change in expression relative to ungerminated conidia ranged between 2.6- and 6-fold. The characterisation of genes involved in biochemical processes during the germination of conidia could be useful for target-specific development of new antifungal agents.

A single nucleotide polymorphism in the wheat powdery mildew (Blumeria graminis f. sp. tritici) cytochrome b gene is responsible for resistance to inhibitors of the quinol outer binding site of the cytochrome bc 1 complex (QoI) fungicides. Analysis of a partial sequence of the cytochrome b gene from field isolates resistant and sensitive to QoI fungicides revealed the same point mutation in barley powdery mildew (B. graminis f. sp. hordei). Analysis of 118 and 40 barley powdery mildew isolates using a cleaved amplified polymorphic sequence assay and denaturing high performance liquid chromatography, respectively, confirmed that this single nucleotide polymorphism also confers resistance to QoI fungicides in barley powdery mildew.

A single nucleotide polymorphism (snp) in the cytochrome b gene confers resistance to strobilurin fungicides in Erysiphe graminis DC f sp tritici Marchal. On the basis of this point mutation three different types of molecular markers have been developed. Cleaved amplified polymorphic sequences and allele-specific PCR were used to score resistant and sensitive isolates from specifically selected regional populations across Europe. The results of molecular tests were in total agreement with the resistance phenotypes revealed by in vivo tests. Serial dilutions of mixed samples (resistant/sensitive) delimited the detection for strobilurin-resistant alleles to a range of 10–50% for both marker classes. Due to these detection limits no mixture of mitochondria within individual isolates was found. Denaturing high performance chromatography was used to increase the detection sensitivity for the mutant allele. Although the detection limit was lowered to 5–10%, there was no evidence for the existence of mixed mitochondrial genotypes. © 2003 Society of Chemical Industry

Pathogenicity data from surveys of Puccinia triticina (formerly P. recondita f. sp. tritici) conducted in western Europe in 1995 were analysed to compare the structure of regional populations of the pathogen. Many of the populations differed in phenotypic diversity and pathotypic composition, even though they occurred within a single epidemiological unit, suggesting that local factors may influence the establishment and propagation of individual pathotypes in the regional populations. Neighbouring regions were more similar than distant regions, and all regions shared at least one pathotype, except populations in northern Italy and Scotland. A high degree of similarity was found between populations in northern France and Great Britain, providing strong evidence of free movement of inoculum between these regions. Resistance genes were postulated for a selection of 91 wheat cultivars, representing those most commonly grown in western Europe in 1995. Thirteen cultivars lacked detectable seedling resistance genes and the remaining 78 possessed from one to three resistance genes; those detected were Lr1, Lr3a, Lr10, Lr13, Lr14a, Lr17b, Lr20, Lr26 and Lr37. The most commonly detected resistance gene was Lr13, which was present singly or in combination with other resistance genes in 48 cultivars (53%). The gene Lr14a was detected in 18 cultivars, Lr26 was present in 16 cultivars. The role of host selection in the composition of the regional populations of P. triticina in western Europe in 1995 was difficult to assess on the basis of the results obtained, since virulence data were not available for Lr13 and Lr14a.

The population structure of Puccinia recondita f. sp. tritici (Prt) in western Europe was examined by assessing variability in pathogenicity and in randomly amplified polymorphic DNA (RAPD) among 61 single uredinial isolates. The isolates were chosen to represent pathotypes detected in a previous survey of pathogenic variability in the fungus in western Europe in 1995. Thirty-five pathotypes were identified by assessing infection types produced by the 61 isolates on 24 differential lines, each with a single gene for resistance to Prt. In contrast, only 18 RAPD phenotypes were identified by scoring 19 polymorphic RAPD bands generated with eight RAPD primers. When analysed by cluster and bootstrap analyses, the pathogenicity and RAPD results revealed little evidence for robust distinct clusters among the isolates. Multiple isolates of several pathotypes collected from widely separated locations such as Belgium, Germany, France, Italy and Switzerland had the same RAPD phenotype, providing evidence of clonal migration over considerable distances in western Europe. Some variability (one or two band differences) was observed in RAPD phenotype within several pathotypes, indicating the possible occurrence of genetic changes independent of pathogenicity, and/or the independent development of pathotypes with different genetic backgrounds. Two groups of isolates identified in the 1995 survey, differentiated by pathogenicity for genes Lr3a, Lr3bg, Lr3ka and Lr30, were not distinguished by RAPD phenotype, indicating that the groups probably do not constitute separate lineages within the pathogen population. Little correlation was apparent between the polymorphisms observed in pathogenicity and RAPD phenotypes. The similarity in the genetic backgrounds of the isolates, as assessed by RAPD markers, suggest that the observed differences in pathogenicity may have arisen by selection for specific virulences corresponding to genes for resistance in wheat cultivars grown in the region. Three isolates of pathotype 3, restricted in its distribution to southern France during 1995, were distinct from all other isolates in RAPD phenotype. Circumstantial evidence suggests that this pathotype originated from northern Africa, and that it belongs to a group of leaf rust pathogens specialized to durum wheats.

A pathogenicity survey of Puccinia recondita f.sp. tritici (Prt) was conducted in western Europe in 1995. Random urediospore isolates (850) of Prt were collected from the air by means of a jet spore sampler in wheat-growing regions of Austria, Belgium, France, Germany, northern Italy, Switzerland and the UK. Pathogenicity of the isolates was determined in tests of detached primary leaf segments maintained on water agar supplemented with benzimidazole (35 p.p.m.). The differential genotypes used were Thatcher, 20 near-isogenic Thatcher lines each with a single leaf rust resistance gene, and five cultivars/lines with additional resistance genes. All isolates were avirulent for the genes Lr9, Lr19, Lr21, Lr24, Lr25 and Lr29, and both virulence and avirulence were detected for the remaining 19 genes. Fifty-three pathotypes were identified, four of which predominated (64% of isolates) and were widespread throughout western Europe. Three of the four predominant pathotypes were also identified in collections of wheat leaf rust collected in Poland, Hungary, Estonia and Finland. One pathotype, which comprised 35% of isolates in the south of France, was not detected in any other region. This pathotype was indistinguishable from several isolates obtained from Morocco, which suggested that it may have originated from northern Africa. Comparisons with previously published data suggested that the four predominant pathotypes were very similar and possibly the same as pathotypes present in the former Czechoslovakia for up to 20 years. The results obtained provide evidence of migration of Prt over considerable distances in western Europe, stressing the need for a co-ordinated approach for genetical control of the disease in this region.

A method is described for establishing isolates of Puccinia recondita f. sp. tritici (causal agent of brown rust of wheat) on detached seedling leaf segments. The method was used to compare the responses of leaf segments and intact seedling leaves for 28 differential genotypes inoculated with eight rust isolates. Leaf segments were incubated at two post-inoculation temperatures (17 and 23C) and intact seedlings at 20–25 C. Reliable determinations of isolate pathogenicity was obtained using detached leaf segments of wheats with genes Lr l. Lr2a, Lr3a, Lr3bg., Lr3ka, Lr9, Lr15, Lrl9. Lr20, Lr24, Lr25. Lr26, Lr28, and Lr30 at both post-inoculation temperatures, and for wheats with genes Lr2b. Lr2c, Lrl7, Lr23, Lr27 + Lr31 and LrH, at 23°C. Differences between leaf segments and intact leaves for the remaining eight differentials were attributed to inconsistent or poor expression of genes in detached leaf segments. By repeating tests with detached leaf segments, it was possible to establish the pathogenicities of the isolates on all of these differentials except those carrying Lr13, Lr14a, Lr16 and Lr18. Potential uses and limitations of the technique in studies of Puccinia recondita f. sp. tritici are discussed. Zusammenfassung Es wird eine Methode beschrieben, die Isolatkultivierung von Puccinia recondita f. sp. tritici(Erreger des Braun-rosts an Weizen) auf Biattsegmenten erlaubt. Mit Hilfe dieser Methode wurde ein Vergleich der Infektions-reaktion zwischen Blattsegmenten und ganzen Pflanzen fur 28 unterschiedliche Genotypen, inokuliert mit acht Braunrostisolaten, vorgenommen. Nach der Inokulation wurden die Blattsegmente bei zwei verschiedenen Temperaturen (17° C und 23°C) und die ganzen Pflanzen bei 20–25°C inkubiert. Eine verlaβliche Bestimmung der Pathogenitat der Isolate war mit der Nutzung von Blattsegmenten bei Weizeniinien mit den Genen Lrl, Lr2a, Lr3a, Lr3bg, LrSka, Lr9, Lr15, Lrl9, Lr20, Lr24, Lr25, Lr26, Lr28, und Lr30 bei beiden Inkubationstemperaturen und fur die Weizeniinien Lr2b, Lr2c, Lrl7, Lr23, Lr27+Lr31 und LrH bti 23°C gegeben. Unterschiede in der Befallsreaktion bei den verbieibenden 8 Linien werden auf eine ungleichmaBige oder schwache Expremierung der Gene in den Blattsegmenten zuruck-gefuhrt. Allerdings war es bei Wiederholung der Tests mit Blattsegmenten moglich, die Pathogenitat der Isolate auf alien Linien mit Ausnahme derer mit den Genen Lr13, Lr14a, Lrl6 und Lrl8 zu bestimmen. Die Nut-zungsmoglichkeiten und Einschrankungen der methode fur Untersuchungen von Puccinia recondita f. sp. Iritici werden diskutiert.

The quantification of single nucleotide polymorphism (SNP) allele frequencies in pooled DNA samples using real time PCR is a promising approach for large-scale diagnostics and genotyping. The limits of detection (LOD) and limits of quantification (LOQ) for mutant SNP alleles are of particular importance for determination of the working range, which, in the case of allele-specific real time PCR, can be limited by the variance of calibration data from serially diluted mutant allele samples as well as by the variance of the 100% wild-type allele samples (blank values). In this study, 3sigma and 10sigma criteria were applied for the calculation of LOD and LOQ values. Alternatively, LOQ was derived from a 20% threshold for the relative standard deviation (%RSD) of measurements by fitting a curve for the relationship between %RSD and copy numbers of the mutant alleles. We found that detection and quantification of mutant alleles were exclusively limited by the variance of calibration data since the estimated LOD(calibration) (696 in 30 000 000 copies, 0.0023%), LOQ(20%RSD) (1470, 0.0049%) and LOQ(calibration) (2319, 0.0077) values were significantly higher than the LOD(blank) (130, 0.0004%) and LOQ(blank) (265, 0.0009%) values derived from measurements of wild-type allele samples. No significant matrix effects of the genomic background DNA on the estimation of LOD and LOQ were found. Furthermore, the impact of large genome sizes and the general application of the procedure for the estimation of LOD and LOQ in quantitative real time PCR diagnostics are discussed.

A single nucleotide polymorphism (snp) in the cytochrome b gene confers resistance to strobilurin fungicides in Erysiphe graminis DC fsp tritici Marchal. On the basis of this point mutation three different types of molecular markers have been developed. Cleaved amplified polymorphic sequences and allele-specific PCR were used to score resistant and sensitive isolates from specifically selected regional populations across Europe. The results of molecular tests were in total agreement with the resistance phenotypes revealed by in vivo tests. Serial dilutions of mixed samples (resistant/sensitive) delimited the detection for strobilurin-resistant alleles to a range of 10-50% for both marker classes. Due to these detection limits no mixture of mitochondria within individual isolates was found. Denaturing high performance chromatography was used to increase the detection sensitivity for the mutant allele. Although the detection limit was lowered to 5-10%, there was no evidence for the existence of mixed mitochondrial genotypes.

Wheat powdery mildew, Blumeria (=Erysiphe) graminis DC f sp tritici Marschal is one of the most important foliar diseases of cereals in Europe, and has shown a high potential for adaptability in sensitivity towards modern fungicides during the last 20 years. Quinoxyfen is a surface-mobile fungicide from a new chemical class that has been commercially used for the control of B graminis in Europe since 1997. When the compound was launched, a Resistance Management Strategy was implemented which included: no seed treatments, no autumn use, a specified window of application and recommendations for appropriate tank-mixing. To assess the success of this Management Strategy, a European-wide resistance monitoring programme was carried out between 1995 and 2000 using spore trap sampling, and a whole plant assay. The mean EC 50 values found by year were 0.060 mg litre -1 in 1995, 0.052 mg litre -1 in 1996, 0.071 ml litre -1 in 1997, 0.039 mg litre -1 in 1998, 0.039 mg litre -1 in 1999 and 0.063 mg litre -1 in 2000. No new sensitivity classes were found. The slight shift to lower sensitivity in 1998 and 1999 was correlated with similar shifts in three wild-type isolates, and was ascribed to experimental variation. The monitoring programme will continue to assess the long-term impact of the management recommendations on the resistance status of quinoxyfen.

Random samples of spores of wheat powdery mildew were collected from the atmosphere by means of a jet spore sampler while driving through selected wheat growing areas in Europe in 1986. Their single colony progenies were tested in the laboratory forvirulence against wheat differential varieties with powdery mildew resistances Pm2, Pm4b, Pm8, Mli and the combinations Pm2+Pm6, Pm4b+Pm8 and Pm2+Pm4b+Pm8. There were regional differences in virulence frequencies. Virulence on Pm2, Pm2+Pm6 and, less expressed, on Pm4b was most frequent in England and decreased mainly to the east, whereas the reverse was true for Pm8. Virulence to Mli was generally high, and to the combined genotypes generally low. The results suggest that large parts of Europe are an epidemiological unit. The data are discussed with respect to selection caused by the varieties grown, as well as to the influence of wind distribution on the composition of the pathogen population. Zusammenfassung Virulenzanalyse von Weizenmehltaupopulationen in Europa Stichproben von Sporen des Weizenmehltaus wurden im Jahre 1986 wahrend der Fahrt durch ausgewahlte Weizenanbaugebiete Europas mit einer Dusensporenfalle aus der Luft gewonnen. Von Nachkommenschaften einzelner Kolonien wurde im Labor die Virulenz gegen die Weizenmehltauresistenzen Pm2, Pm4b, Pm8 und Mli sowie die Resistenzkombinationen Pm2+Pm6, Pm4b+Pm8 und Pm2+Pm4b+Pm8 bestimmt. Die Haufigkeiten der Virulenzeigenschaften waren regional differenziert. Virulenz gegen Pm2, Pm2+Pm6 und, weniger ausgepragt, gegen Pm4b war in England am haufigsten und nahm in ostwartiger Richtung ab, wahrend das Gegenteil fur Pm8 der Fall war. Virulenz gegen Mli wurde generell haufig gefunden und gegen kombiniene Genotypen selten. Nach den Ergebnissen miissen weite Gebiete Europas als epidemiologische Einheit angesehen werden. Die Zusammensetzung der Erregerpopulation wird diskuiien im Zusammenhang mit der Selektion auf Grund der Sortennutzung zum einen wie auch dem Einflus der Windverbreitung zum anderen.

An instrument for in-the-field testing of citrus fruit firmness has been developed and tested. Linear deformation of a fruit along its diameter under the pressure of a constant force was found a good criterion of weight loss, and hence firmness of citrus.

We discuss criteria for the design of cold storage facilities for use in research. Factors considered are: determination of the desired accuracy of temperature and relative humidity control, methods of air circulation and fresh air introduction.

An inexpensive method for accurate control and measurement of fresh air introduction into experimental storage rooms is described.