Pathogenicity of Rhynchosporium secalis isolates from Norway on 30 cultivars of barley

The pathogenic variability of the barley scald fungus, Rhynchosporium secalis , in central Norway was examined in 1994. The climate in this region is usually cold and wet during the growing season of spring barley. Leaf blotch is prevalent and causes significant yield losses. Forty-two isolates of the fungus, from naturally infected spring barley in four counties, were differentiated into 32 pathotypes by the standard differential set for R. secalis . All pathotypes were complex and had virulence for nine to 22 differentials. The cultivar Osiris was resistant to all isolates tested. The cultivars C.I.8162, Hudson, Atlas 46 and C.I.3515 were resistant to the majority of the isolates. Several differentials with various resistance genes were susceptible to up to 100% of the isolates. Isolates were derived from local cultivars with no known resistance genes, suggesting that R. secalis populations in central Norway are characterized by a high degree of seemingly unnecessary pathogenicity. Because of the great variability and complexity of the pathotypes, traditional breeding methods using single major genes are not likely to be effective in central Norway.     

Application of real-time and multiplex polymerase chain reaction assays to study leaf blotch epidemics in barley

ABSTRACT Leaf blotch, caused by Rhynchosporium secalis, was studied in a range of winter barley cultivars using a combination of traditional plant pathological techniques and newly developed multiplex and real-time polymerase chain reaction (PCR) assays. Using PCR, symptomless leaf blotch colonization was shown to occur throughout the growing season in the resistant winter barley cv. Leonie. The dynamics of colonization throughout the growing season were similar in both Leonie and Vertige, a susceptible cultivar. However, pathogen DNA levels were approximately 10-fold higher in the susceptible cultivar, which expressed symptoms throughout the growing season. Visual assessments and PCR also were used to determine levels of R. secalis colonization and infection in samples from a field experiment used to test a range of winter barley cultivars with different levels of leaf blotch resistance. The correlation between the PCR and visual assessment data was better at higher infection levels (R(2) = 0.81 for leaf samples with >0.3% disease). Although resistance ratings did not correlate well with levels of disease for all cultivars tested, low levels of infection were observed in the cultivar with the highest resistance rating and high levels of infection in the cultivar with the lowest resistance rating.     

Asexual Genetic Exchange in the Barley Pathogen Rhynchosporium secalis

ABSTRACT The causal agent of barley scald, Rhynchosporium secalis, is a haploid anamorphic ascomycete with no known sexual stage. Nevertheless, a high degree of genetic variation has been observed in fungal populations on commercial barley cultivars and parasexuality has been suggested to contribute to this variation. In order to test whether asexual genetic exchange can occur, isolates of R. secalis were transformed to hygromycin B resistance or phleomycin resistance. Mixtures of transformants were co-inoculated either on agar or in planta and screened for the occurrence of dual-antibiotic-resistant colonies. No dual-antibiotic-resistant colonies resulted from mixing transformants of different fungal isolates. In contrast, with transformants originating from the same fungal isolate, asexual exchange of markers was demonstrated on agar plates and in planta. This is the first definitive evidence of asexual genetic exchange in R. secalis.     

Rhynchosporium leaf scald disease incidence: seed source and spatial pattern

A programme of field trials for the study of the winter barley–Rhynchosporium commune pathosystem is reported. The associated seedborne disease rhynchosporium leaf scald is regarded as having an important impact on barley yields. The analysis in this study relates to the impact of the seed source (commercial or farm-saved seed) on disease incidence and to the spatial pattern of rhynchosporium leaf scald disease incidence. Disease incidence data were calculated from field data recorded as disease severity. Mean disease incidence was higher in the crops grown from farm-saved seed than in those grown from commercial seed, although great agronomic significance cannot be attached to this result. The spatial pattern of rhynchosporium leaf scald disease incidence was characterized in terms of the binary power law (BPL) and was indicative of an aggregated pattern. Programme-wide BPL results were described using a novel phytopathological application of a random coefficients model. These results have application in field sampling for rhynchosporium leaf scald disease.     

Infection of Rrs1 barley by an incompatible race of the fungus Rhynchosporium secalis expressing the green fluorescent protein

Scald disease of barley, caused by the fungal pathogen Rhynchosporium secalis, is one of the most serious diseases of this crop worldwide. Disease control is achieved in part by deployment of major resistance (Rrs) genes in barley. However, in both susceptible and resistant barley plants, R. secalis is able to complete a symptomless infection cycle. To examine the R. secalis infection cycle, Agrobacterium tumefaciens‐mediated transformation was used to generate R. secalis isolates expressing the green fluorescent protein or DsRed fluorescent protein, and that were virulent on an Rrs2 plant (cv. Atlas), but avirulent on an Rrs1 plant (cv. Atlas 46). Confocal laser scanning microscopy revealed that R. secalis infected the susceptible cultivar and formed an extensive hyphal network that followed the anticlinal cell walls of epidermal cells. In the resistant cultivar, hyphal development was more restricted and random in direction of growth. In contrast to earlier models of R. secalis infection, epidermal collapse was not observed until approximately 10 days post‐inoculation in both cultivars. Sporulation of R. secalis was observed in both susceptible and resistant interactions. Observations made using the GFP‐expressing isolate were complemented and confirmed using a combination of the fluorescent probes 5‐chloromethylfluorescein diacetate and propidium iodide, in the non‐transformed wild‐type isolate. The findings will enable the different Rrs genes to be better characterized in the effect they exert on pathogen growth and may aid in identification of the most effective resistance.     

Variation amongst isozymes of Rhynchosporium secalis

Mycelial extracts of 288 single-spore isolates of Rhynchosporium secalis. derived from infected leaves collected from seven barley cultivars at nine sampling sites, were subjected to horizontal slab polyacrylamide gel electrophoresis. A total of 28 patterns for α-esterase, four for β-glucosidase and one for β-galactosidase were obtained. One probable isozyme polymorphism, for α-esterase, was identified. There was no correlation between any particular band or pattern and either host cultivar or site.     

Evidence of asexual recombination in Rhynchosporium secalis

Three single-spore isolates of Rhynchosporium secalis that differed in their α-esterase and β-glucosidase isozyme patterns were inoculated as two mixtures, each of two isolates, on to seedlings of the susceptible barley cultivar Maris Mink. Approximately 100 single-spore isolates were taken from mature lesions produced by each mixture. These were subjected to polyacrylamide gel electrophoresis and the gels were stained for α-esterase and β-glucosidase. Parental types only were produced by one of the isolate mixtures. However, one of the 10 lesions examined for the second mixture produced nine isolates with a novel combination of isozymes, indicating that some form of asexual recombination had occurred. The use of isozymes as a natural marker system for the detection in vivo of asexual recombination in pathogenic fungi is discussed.     

Associations between fungal and abiotic leaf spotting and the presence of mlo alleles in barley

The hypothesis that the increased use of the powdery mildew-resistance gene mlo has caused the increase in spotting diseases of barley over the past 20 years was tested in field trials. Near-isogenic lines with alleles of the Mlo gene for susceptibility or resistance to mildew in two parental backgrounds were trialled at four sites in Scotland and two in Ireland that were prone to spotting diseases, over 3 consecutive years. Mildew was controlled by sprays with quinoxyfen. Disease levels were low in the trials, the two most important diseases being scald caused by Rhynchosporium secalis and ramularia leaf spot caused by Ramularia collo-cygni . There were high levels of abiotic spotting. Lines with mutant mlo alleles consistently developed less Rh. secalis and Ra. collo-cygni , but more abiotic spots. This study indicates that the mlo mildew-resistance gene has not alone been responsible for the rise in spotting diseases over the past 20 years. Possible reasons for the rise are discussed, including the interaction of the mlo gene with the environment.     

Effect of barley yellow dwarf virus on susceptibility of barley cultivars to net blotch (Pyrenophora teres) and leaf blotch (Rhynchosporium secalis)

The effect of barley yellow dwarf virus (BYDV) on the development of net blotch (Pyrenophora eves) and leaf blotch (Rhynchosporium secalis) was examined on seven barley cuitivars. Seedlings were infected with BYDV at the two-leaf stage (G.S. 12). Their susceptibility to three isolates of p teres and isolates of two races (U.K. 1, U.K. 2) of R. secalis was examined at the four-leaf stsge (G.S. 14) and when plants were more mature (G.S. 33/38). At G.S. 14 numbers of lesions produced by P. teres and R. secalis were reduced, on average, by 37 and 72% respectively, and at G.S. 33/38 by 61 and 74%.     

Resistance to Scald (Rhynchosporium secalis) in Barley (Hordeum vulgare) Studied by Near-Isogenic Lines: I. Markers and Differential Isolates

ABSTRACT Near-isogenic lines (NILs) with resistance for scald in seventh generation backcross with 'Ingrid' as recurrent parent (RP) were tested with seven differential isolates of Rhynchosporium secalis in Norway and Canada. NILs of 'Turk', 'Brier', 'CI 8162', 'La Mesita', 'Hispont', 'Atlas 46', 'Modoc', 'Hudson', 'Abyssinian', 'Steudelli', and 'CI 2222' also were evaluated for field reactions. The genetic characterization of the NILs (degree of isogeneity with Ingrid) and with each other was carried out. The molecular marker pattern shows that the backcrossing program has resulted in from 86.3 to 100% RP genome in the NILs, depending on the marker system. On an average, 96% RP genome was found in the NILs. There were certain consistent (pairwise) differences between the NILs and RP on chromosomes 3H and 7H. Both chromosomes are known to contain loci conferring resistance to R. secalis, indicating successful introgression from the donors into the NILs. Approximately two-thirds of the observed RP-NIL polymorphisms were linked to the assumed resistance in the NIL. Based on the marker and phenotypic analyses of the NILs, suggestions for a more appropriate and updated terminology of genes for resistance to R. secalis in barley are made. The proposed changes in nomenclature also indicate the differentials that are available as NILs and those lacking.     

Quantification of seedborne infection by Rhynchosporium secalis in barley using competitive PCR

The potential of the competitive polymerase chain reaction (PCR) assay for quantification of seedborne infection by Rhynchosporium secalis in barley was examined using a primer set (RS1 and RS3) derived from the internal transcribed spacer (ITS) regions of ribosomal RNA genes of this pathogen. Introduction of a heterologous internal control, which competes for the same primer set in the conventional PCR assay, allowed for detection and quantification of R. secalis fungal biomass. In order to generate a standard calibration curve, DNA prepared from infected seeds with different levels of R. secalis infection was subjected to competitive PCR assay. The resulting PCR product ratio for each PCR reaction ( R. secalis -amplified DNA/internal control template-amplified DNA) increased proportionally with increasing levels of infected seed DNA in the reaction mixture. Naturally infected seed lots collected from 1995 to 1999 were used to demonstrate the potential of the competitive PCR assay as an alternative seed health testing method. The results from this competitive PCR assay were compared with those from conventional visual disease assessment and an agar plate assay. Although relatively good correlation between visual disease assessment and the competitive PCR was found in the case of artificially mixed seed samples, there was poor correlation in the experiments using naturally infected seed samples.     

The role of seeds and airborne inoculum in the initiation of leaf blotch (Rhynchosporium secalis) epidemics in winter barley

Both airborne spores of Rhynchosporium secalis and seed infection have been implied as major sources of primary inoculum for barley leaf blotch (scald) epidemics in fields without previous history of barley cropping. However, little is known about their relative importance in the onset of disease. Results from both quantitative real‐time PCR and visual assessments indicated that seed infection was the main source of inoculum in the field trial conducted in this study. Glasshouse studies established that the pathogen can be transmitted from infected seeds into roots, shoots and leaves without causing symptoms. Plants in the field trial remained symptomless for approximately four months before symptoms were observed in the crop. Covering the crop during part of the growing season was shown to prevent pathogen growth, despite the use of infected seed, indicating that changes in the physiological condition of the plant and/or environmental conditions may trigger disease development. However, once the disease appeared in the field it quickly became uniform throughout the cropping area. Only small amounts of R. secalis DNA were measured in 24 h spore‐trap tape samples using PCR. Inoculum levels equivalent to spore concentrations between 30 and 60 spores per m³ of air were only detected on three occasions during the growing season. The temporal pattern and level of detection of R. secalis DNA in spore tape samples indicated that airborne inoculum was limited and most likely represented rain‐splashed conidia rather than putative ascospores.     

Genetic Structure of Rhynchosporium secalis in Australia

ABSTRACT Restriction fragment length polymorphism (RFLP) markers were used to determine the genetic structure of Australian field populations of the barley scald pathogen Rhynchosporium secalis. Fungal isolates were collected by hierarchical sampling from five naturally infected barley fields in different geographic locations during a single growing season. Genetic variation was high in Australian R. secalis populations. Among the 265 fungal isolates analyzed, 214 distinct genotypes were identified. Average genotype diversity within a field population was 65% of its theoretical maximum. Nei's average gene diversity across seven RFLP loci was 0.54. The majority (76%) of gene diversity was distributed within sampling site areas measuring 1 m(2); 19% of gene diversity was distributed among sampling sites within fields; and 5% of gene diversity was distributed among fields. Fungal populations from different locations differed significantly both in allele frequencies and genotype diversities. The degree of genetic differentiation was significantly correlated with geographic distance between populations. Our results suggest that the R. secalis population in Western Australia has a different genetic structure than populations in Victoria and South Australia.     

The effect of component number on Rhynchosporium secalis infection and yield in mixtures of winter barley cultivars

Mixtures of winter barley cultivars containing up to six components were grown over three years with and without fungicide treatment. Yield increases were recorded for mixtures compared with the mean of their monoculture components and there was a significant trend towards greater benefit from increased number of components. These benefits were partially attributable to a corresponding increase in control of Rhynchosporium secalis as component number increased. The potential for exploitation of mixtures in cereals for control of splash-dispersed pathogens is discussed.     

Leaf blotch severity on spring barley infected by isolates of Rhynchosporium secalis under different temperature and humidity regimes

The infection efficiency and severity of leaf blotch on spring barley inoculated with three pathotypes of Rhynchosporium secalis from central Norway were studied under different temperature and humidity regimes. Seedlings of the cultivar Arve were subjected to two constant temperatures, 13° or 18°C. Dry periods of 8 h or longer before or after a wet period of 4 h, carried out in the first 48 h postinoculation, reduced disease severity assessed 16 days after inoculation. The effect of dry periods of up to 24 h was nullified when plants were subjected to high humidity for 48 h after the dry treatment. The disease developed most rapidly when the wet period was 48 h and the temperature 18°C. At or near the optimum temperature for R. secalis (18°C), leaf wetness duration as short as 2 h resulted in considerable disease. Isolates reacted differently to temperature. The most aggressive isolate caused severe disease irrespective of temperature (56–70% of the leaf area infected); however, disease severity caused by the least aggressive isolate was significantly higher at the optimum temperature compared with a lower temperature (13°C). This information can facilitate evaluation of weather data in relation to predicting leaf blotch for advisory purposes.     

Differential Selection on Rhynchosporium secalis During Parasitic and Saprophytic Phases in the Barley Scald Disease Cycle

ABSTRACT Competition among eight Rhynchosporium secalis isolates was assessed during parasitic and saprophytic phases of the disease cycle in field experiments conducted at two locations and over two growing seasons. The eight isolates were inoculated onto six barley populations exhibiting varying degrees of resistance. Microsatellite analysis of 2,866 isolates recovered from the field experiments showed significant, and sometimes opposite, changes in the frequencies of R. secalis genotypes during the growing season (parasitic phase) and between growing seasons (saprophytic phase). Isolates that showed the most complex virulence in greenhouse seedling assays had the lowest fitness in the field experiment. Significant differences in isolate fitness were found on different host populations and in different environments. Selection coefficients were large, indicating that evolution can occur rapidly in field populations. Although inoculated isolates had the lowest overall fitness on the moderately resistant landrace cv. Arabi Aswad, some isolates were more virulent and consistently increased in frequency on this landrace, suggesting a risk of directional selection and possible erosion of the resistance following its widespread deployment in monoculture. These results provide the first direct evidence that R. secalis pathogen genotypes differ in their saprophytic ability and parasitic fitness under field conditions.     

Virulence spectrum to barley (Hordeum vulgare L.) in isolates of Rhynchosporium secalis from Syria

Journal of Plant Diseases and Protection - Scald caused by Rhynchosporium secalis, is an economically important disease found worldwide. Information on the variability of virulence in R. secalis is...     

Characterisation of BaYMV and BaMMV Pathotypes in France

The reaction of thirty-four barley cultivars from European and Asiatic origin was analysed in six soils infected with barley yellow mosaic virus complex (BaYMV, BaMMV). These soils were selected from 16 sites for their differences in cultivar response. Amongst the six cultivars carrying the ym4 gene (Esterel, Express, Labéa, Majestic, Réjane, Vanoise), only cv Majestic was infected at one site with BaYMV and BaMMV. Concerning BaYMV, three cultivars were infected on all the soils and 19 on none of them. Twelve cultivars were differentially infected depending on the soil. In the case of BaMMV, four cultivars were infected on all the soils and 19 on none of them. Eleven cultivars were differentially infected depending on the soil. ELISA tests revealed the presence, in these soils, of variants of BaYMV and BaMMV that were able to overcome at least seven of the 12 known resistance genes (ym3, ym4, ym6, ym8, ym9, ym10, ym11) and the resistance of three varieties (Tosan Kawa 73, OU1 and Taihoku A) in which the genetic basis is unknown. Amplification by RT-PCR of the N-terminal region for three of BaYMV variants was performed. Nucleotide and amino acid sequences were determined and compared with the corresponding sequence of a common strain of BaYMV-G. A few nucleotide differences were detected between all the French isolates, but there were no strain specific amino acid differences.     

The occurrence of carbendazim resistance in Rhynchosporium secalis on winter barley in England and Wales in 1992 and 1993

In 1992, samples of Rhynchosporium secalis from 19 winter barley crops in England and Wales were examined for carbendazim resistance. Of the 120 isolates obtained, 14·2% were resistant to carbendazim. A larger survey was carried out in England and Wales in 1993 when samples from 74 crops were examined. On this occasion 16·6% of the 639 isolates obtained were resistant to carbendazim, and resistant isolates were detected in 46% of crops.     

Global Hierarchical Gene Diversity Analysis Suggests the Fertile Crescent Is Not the Center of Origin of the Barley Scald Pathogen Rhynchosporium secalis

A total of 1,366 Rhynchosporium secalis isolates causing scald on barley, rye, and wild barley (Hordeum spontaneum) were assayed for restriction fragment length polymorphism loci, DNA fingerprints, and mating type, to characterize global genetic structure. The isolates originated from 31 field populations on five continents. Hierarchical analysis revealed that more than 70% of the total genetic variation within regions was distributed within a barley field. At the global level, only 58% of the total genetic variation was distributed within fields, while 11% was distributed among fields within regions, and 31% was distributed among regions. A significant correlation was found between genetic and geographic distance. These findings suggest that gene flow is common at the local level while it is low between regions on the same continent, and rare between continents. Analyses of multilocus associations, genotype diversity, and mating type frequencies indicate that sexual recombination is occurring in most of the populations. We found the highest allele richness in Scandinavia followed by Switzerland. This suggests that R. secalis may not have originated at the center of origin of barley, the Fertile Crescent, nor in a secondary center of diversity of barley, Ethiopia.