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.     

Somatic recombination in Rhynchosporium secalis

Drug-resistant mutants were induced in strains of Rhynchosporium secalis. Two mutants resistant to hygromycin-B and 5-fluorouracil, respectively, were stable and easily selected in culture. Mixtures of the two mutants failed to yield any recombinants, whether under selection pressure or not, on agar and on barley leaves. After protoplast production and fusion, followed by selection on media containing both drugs, two recombinants were isolated. Single-spore derivatives expressed the drug resistances of both parental strains.     

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.     

Characterisation of benzimidazole-resistant strains of Rhynchosporium secalis

Abstract: Benzimidazole-resistant mutants of Rhynchosporium secalis were easily generated in the laboratory using UV mutagenesis. Three levels of resistance were identified (low, LR; moderate, MR; high, HR), but there was no negative cross-resistance with N-phenylcarbamate fungicides. In all cases pathogenicity was reduced, in some cases drastically. Benzimidazole-resistant field strains were first detected in 1990, some 15 years after this fungicide group was first used in UK barley crops. Unlike laboratory mutants, only HR phenotypes were isolated from the field, and all showed negative cross-resistance to N-phenylcarbamates. These field resistant strains were no less pathogenic than wild-type ones. Carbendazim binding to tubulin-like protein from HR phenotypes, whether generated in the laboratory or isolated from the field, was reduced.     

The Genetic Structure of Field Populations of Rhynchosporium secalis from Three Continents Suggests Moderate Gene Flow and Regular Recombination

ABSTRACT Restriction fragment length polymorphism (RFLP) markers were used to compare the genetic structure of field populations of Rhynchosporium secalis from barley. A total of 543 isolates representing 8 field populations were sampled from Australia, California, Finland, and Norway. Gene and genotype diversity were high in all populations. Nei's average gene diversity across seven RFLP loci was 0.513. Hierarchical gene diversity analysis showed that 9% of the total genetic variability was distributed among continents, 4% was distributed among fields within continents, and 13% was distributed among collection stations within a field. The majority (74%) of genetic variability was distributed within collection areas of approximately 1 m(2) within fields. Gene flow appears to be significant on a regional scale but more restricted among continents. Allele frequencies were significantly different at several RFLP loci. Genetic distances were small among populations within regions and large between regions. Pairwise comparisons of genotype diversity in the populations revealed significant differences among populations that were related mainly to differences in sampling strategies. Isolates from Norway and Finland showed a lower copy hybridization pattern with probe pRS26. This probe functioned as a fingerprint probe for the California and Australian isolates. Seven out of the eight populations studied were at gametic equilibrium for RFLP loci, suggesting that R. secalis populations in Norway, Finland, and Australia undergo regular recombination, although a teleomorph has not yet been recognized.     

Histopathological Study of Barley Cultivars Resistant and Susceptible to Rhynchosporium secalis

ABSTRACT Differences in the penetration process by Rhynchosporium secalis were compared in resistant and susceptible barley cultivars at the seedling stage. Percent penetration and percent host cell wall alteration (HCWA) differed significantly among cultivars and isolates as revealed by light microscopy. Based on these two variables, the cultivars were statistically separated into two groups that corresponded to their disease reactions. The resistant cultivars, Johnston and CDC Guardian, showed 81.2 to 99.4% HCWA and 0.1 to 20.1% penetration at encounter sites, whereas the susceptible cultivars, Harrington, Argyle, and Manley, had 30.1 to 78.3% HCWA and 31.8 to 81.8% penetration. In the current study, cv. Leduc, which is susceptible at the seedling stage and resistant at the adult stage, showed the same percent HCWA and penetration as did susceptible cultivars. A significant negative correlation (P < 0.01) was found between percent penetration and percent HCWA for cultivars inoculated with two isolates of the pathogen. Isolate 1 was less virulent than isolate 2 with respect to percent penetration and induced significantly fewer HCWA. Scanning electron microscopy showed various shapes of fungal appressoria but no apparent difference in host reaction between resistant and susceptible cultivars. Transmission electron microscopy revealed interactions between the host and pathogen at various stages of penetration. The resistant cv. Johnston responded by producing appositions, as evidenced by a layer of compact osmiophilic material deposited on the inner side of the cell wall. Infection pegs produced by conidia were unable to penetrate the cuticle where an apposition had formed inside. When penetration occurred in the susceptible cv. Argyle, cytoplasmic aggregates and separation of the plasmalemma were visible from the host cell wall, but the layer of compact osmiophilic material was not always present. Data based on light microscopic observations suggested that HCWA may be one of the mechanisms responsible for resistance that is characterized as penetration prevention rather than as a slow rate of mycelial growth after successful penetration. HCWA occurred in response to attempted cuticle penetration, suggesting that HCWA may produce chemical barriers that help to prevent penetration.     

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.     

Using allele-specific oligonucleotide probes to characterize benzimidazole resistance in Rhynchosporium secalis

Benzimidazole fungicides are important mixture components in strategies to combat fungicide resistance in Rhynchosporium secalis Davis. To monitor the performance of these strategies, a rapid, accurate assay has been developed to detect point mutations in the β-tubulin gene which confers resistance of benzimidazoles. The β-tubulin gene of a benzimidazole-resistant strain of R. secalis has been cloned and sequenced. Except for the difference in the position of one of its six introns, this gene showed a strong homology with other β-tubulin genes from filamentous fungi. Resistance was related to a point mutation in codon 198 which caused a glutamic acid to glycine change in resistant field strains, but glutamic acid to lysine in a laboratory mutant. A DNA fragment surrounding codon 198 was amplified directly from diseased lesions using a ‘nested’ set of PCR primers. Combining PCR amplificiation of a target DNA sequence with hybridization of Allele-Specific Oligonucleotide probes (ASO_s, 15-mers) allowed accurate detection of benzimidazole resistance. Only two probes, one sensitive and one resistant, were sufficient to monitor current field populations. Detection was achieved using either ³²P-labelled probe, or non-radioactively using a biotin-labelled probe coupled to streptavidin/alkaline phosphatase. This rapid method using ASOS can detect benzimidazole resistance within 48 h compared with 6–8 weeks by conventional assay procedures.     

Resistance, epidemiology and sustainable management of Rhynchosporium secalis populations on barley

Rhynchosporium secalis is one of the most destructive pathogens of barley worldwide, causing yield decreases of up to 40% and reduced grain quality. Rhynchosporium is a polycyclic disease. Primary inoculum includes conidia produced on crop debris, infected seeds and possibly ascospores, although these have not yet been identified. Secondary disease spread is primarily by splash dispersal of conidia produced on infected leaves, which may be symptomless early in the growing season. Host resistance to R. secalis is mediated by both 'major' or host-specific genes (complete resistance) and 'minor' genes of smaller, generally additive effects (partial resistance). Crop growth stage and plant or canopy architecture can modify the expression of resistance. Resistance genes are distributed unevenly across the barley genome, with most being clustered on the short arms of chromosomes 1H, 3H, 6H and 7H, or in the centromeric region or on the long arm of chromosome 3H. Strategies used to manage rhynchosporium epidemics include cultivar resistance and fungicides, and also cultural practices such as crop rotation, cultivar mixtures and manipulation of sowing date, sowing rate or fertiliser rate. However, the high genetic variability of R. secalis can result in rapid adaptation of pathogen populations to render some of these control strategies ineffective when they are used alone. Sustainable control of rhynchosporium needs to integrate major-gene-mediated resistance, partial resistance and other strategies such as customized fungicide programmes, species or cultivar rotation, resistance gene deployment, clean seed and cultivar mixtures.     

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.     

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.     

Effects of Rhynchosporium secalis at specific growth stages on barley grown in a controlled environment

Under controlled environmental conditions, inoculation of barley at five growth stages with Rhynchosporium secalis reduced grain yield by up to 30%. Greater yield losses resulted from inoculations carried out after tillering than from those during or before tillering. Most of the yield loss was due to a reduction in the number of heads per plant. The number of grains per head was reduced only when plants were inoculated at mid- or late-tillering growth stages. The mean weight of individual grains was not reduced significantly by any treatment. Inoculation at stem elongation also resulted in less leaf area and water use, and delayed anthesis. Inoculation affected root dry weight more than shoot dry weight and all inoculations reduced root dry weight at the boot growth stage     

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...     

A laboratory assay to determine the sensitivity of Rhynchosporium secalis to the fungicide triadimenol

Thirty single-spore isolates of Rhynchosporium secalis were assayed in vitro to assess relative sensitivities to the fungicide triadimenol. Minimum inhibitory concentrations ranged from 0.2 to 3.2 μg/ml. Isolates collected in 1981 from winter barley crops treated with triadimenol + tuberidazole, triadimefon, or prochloraz were as sensitive as those from untreated crops, and four isolates collected prior to the widespread use of these fungicides were not especially sensitive. All isolates tested in the greenhouse on winter and spring barley were controlled by Baytan (triadimenol and fuberidazole).     

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.