The relationship between leaf blotch caused by Rhynchosporium secalis and losses in grain yield of spring barley

Two methods were used to investigate the loss in grain yield associated with specific levels of leaf blotch. Yields from plots sprayed with fungicide were compared with those from unsprayed plots and yields of varieties of different susceptibility to the disease were compared with one another. A disease assessment key is presented, which was used to assess the percentage laminar area of the top two leaves affected by the disease. A linear relationship between disease on the upper two leaves and yield was established. Results from nine trials showed a consistent relationship between the disease level, at growth stage 11·1 (Feekes scale), and loss in yield. The loss in yield expressed as a percentage of the yield of an uninfected crop was equivalent to approximately two-thirds of the percentage of the flag-leaf area visibly infected, or one-half of the infected area on the second leaf. The predicted loss in yield is the average of these two estimates.     

Seasonal changes in primary and secondary inoculum during epidemics of leaf blotch (Rhynchosporium secalis) on winter barley

Seasonal changes in numbers of conidia of Rhynchosporium secalis on debris from previous barley crops infected with leaf blotch (primary inoculum) were monitored in 1985–86 and 1986–87. In 1986–87, changes in numbers of conidia on leaves of plants in the new winter barley crop (secondary inoculum) were also recorded. The greatest increases in production of primary inoculum were in early spring after rain, when temperatures were increasing after periods of sub-zero temperatures when there was little conidial production. Subsequently, more conidia were recovered from this debris after cycles of drying and rewetting than when it remained wet. After January 1987, amounts of secondary inoculum produced on the crop were much greater than amounts of primary inoculum on debris. Most spores were produced on the basal leaves and more spores were present on the September-sown than on the November-sown crop. Thus, while primary inoculum was a source of disease when plants were emerging, secondary inoculum on basal leaves was the main source of disease at stem extension, especially on early-sown crops.     

Effects of fungicide timing on control of Rhynchosporium secalis in barley plants

Sprays of captafol, carbendazim, carbendazim + tridemorph + maneb, diclobutrazol, triadimefon or triadimefon + carbendazim all completely protected barley plants in a glasshouse against R. secalis for at least 30 days. However, their effectiveness in preventing disease development when applied after inoculation differed: triadimefon, traidimefon + carbendazim, or diclobutrazol were the most effective, completely preventing symptom development when applied up to 5 days after inoculation to plants grown above 16 °C, and up to 8 days below 8 °C. All the fungicides decreased the number of viable conidia produced by leaf blotch lesions, and when applied to infected plants at G. S. 30, greatly decreased the upward spread of the disease under simulated rain conditions; the most effective fungicides in these respects were triadimefon and triadimefon + carbendazim. The above fungicides and fungicide mixtures, together with the recently introduced materials fenpropimorph and propiconazole were applied to diseased winter barley crops in winter or in spring. All treatments decreased leaf blotch development and increased yields. In most cases, a winter application was more effective than spring applications, particularly if applied in mid-November. The most effective fungicides were triadimefon and propiconazole. The field trials data fitted well with the predictions of performance indicated by the glasshouse investigations.     

Development of sequence tagged microsatellites (STMs) for the barley scald pathogen Rhynchosporium secalis

A rapid and cost efficient technique was developed and used to generate 168 sequence tagged microsatellites (STMs) in the barley scald pathogen Rhynchosporium secalis. Sixty‐two STMs, amplifying 66 loci, revealed a high level of polymorphism among a diverse set of 16 Australian isolates. Each locus revealed two to nine alleles (average 4 ± 1.82), and a gene diversity measure of 0.54 was obtained. This technique not only halved the cost of marker development compared to traditional methods, but substantially reduced the cost of performing fluorescence‐based microsatellite assays. These STMs provide a powerful tool for genetic studies in R. secalis.     

The origin and colonization history of the barley scald pathogen Rhynchosporium secalis

The origins of pathogens and their past and present migration patterns are often unknown. We used phylogenetic haplotype clustering in conjunction with model-based coalescent approaches to reconstruct the genetic history of the barley leaf pathogen Rhynchosporium secalis using the avirulence gene NIP1 and its flanking regions. Our results falsify the hypothesis that R. secalis emerged in association with its host during the domestication of barley 10,000 to 15,000 years ago in the Fertile Crescent and was introduced into Europe through the migration of Neolithic farmers. Estimates of time since most recent common ancestor (2500-5000 BP) placed the emergence of R. secalis clearly after the domestication of barley. We propose that modern populations of R. secalis originated in northern Europe following a host switch, most probably from a wild grass onto cultivated barley shortly after barley was introduced into northern Europe. R. secalis subsequently spread southwards into already established European barley-growing areas.     

RFLP markers linked to scald (Rhynchosporium secalis) resistance gene Rh2 in barley

Rhynchosporium secalis is the causal organism of barley scald disease. A number of resistance genes against the fungus are well known; one of them, the single dominant Rh2 resistance gene, has been mapped on the linkage map of barley using RFLP (restriction fragment length polymorphism) markers. The Rh2 gene was located on the distal part of chromosome arm 1S co-segregating with the RFLP marker CDO545 in 85 doubled-haploid progeny plants. The spring barley test population used was a cross between the 6-rowed American spring barley cv Atlas, C.I. 4118, carrying the Rh2 resistance gene, and a Bavarian 2-rowed malting barley cv Steffi, susceptible for R. secalis. The assessment of resistance versus susceptibility was based on artificial infections with a one-spore inoculum in greenhouse tests and with pathotype mixtures in field tests. By testing a pathotype mixture of German origin good resistance was found for the Rh2 gene in the field.     

Isolation and characterization of microsatellite loci from the barley scald pathogen,Rhynchosporium secalis

Fifteen primer pairs were designed for 14 polymorphic microsatellite loci, which were isolated and characterized from genomic libraries of Rhynchosporium secalis. Conditions for multiplexing and simultaneous genotyping of up to eight loci in a single run are described. The number of alleles per polymorphic locus ranged from two to 13 in populations from Switzerland and Australia. Genotypic diversity ranged from 61.5 to 66.7. Gene diversity ranged from 0.08 to 0.89 for individual polymorphic loci, with averages of 0.54 and 0.62 for the populations from Switzerland and Australia, respectively. Variable levels of polymorphism make these ideal markers for population genetic analyses.     

The effect of three herbicides on the number of spores of Rhynchosporium secalis on barley stubble and volunteer plants

Single sprays of paraquat, glyphosate or dinoseb-in-oil, were applied to barley stubble or volunteer plants and their effects on the number of spores of Rhynchosporium secalis were studied weekly. Spore concentrations were measured by washing samples of stubble or volunteer plants and counting spores using a haemocytometer. The number of spores recovered from stubbles was relatively small; both paraquat and glyphosate caused some reductions but the results were not consistent. Treatment with paraquat generally caused a transitory increase on volunteer plants; this effect was not found after glyphosate. Both herbicides caused persisting reductions from 4 wk after treatment. Volunteer plants treated with dinoseb-in-oil had fewer spores for 6 wk after treatment, but the plants were not killed and when new growth became infected, spore production recovered to equal that on untreated plants. The fungicide captafol caused some reductions in spore number on stubble but results were not consistent, applied after paraquat it had no effect. It was generally ineffective when applied to volunteer plants; applied after paraquat it tended to increase spore number, but the differences were seldom significant. Spores washed from volunteer plants treated with paraquat or glyphosate were inoculated to pot-grown barley seedlings; neither herbicide had any effect on spore viability. Viable spores were recovered in February from stubble or volunteer plants treated the previous autumn with paraquat or glyphosate respectively.     

Phylogeographical analyses reveal global migration patterns of the barley scald pathogen Rhynchosporium secalis

A phylogeographical analysis of the scald pathogen Rhynchosporium secalis was conducted using nuclear DNA sequences from two neutral restriction fragment length polymorphism loci and the mating-type idiomorphs. Approximately 500 isolates sampled from more than 60 field populations from five continents were analysed to infer migration patterns and the demographic history of the fungus. Migration rates among continents were generally low, consistent with earlier reports of significant population subdivision among continents. Northern Europe was mainly a source population for global migration. We hypothesize that the pathogen only recently moved out of its centre of origin, resulting in founder populations that are reproductively isolated due to the contemporary absence of long-distance gene flow.     

New insights into the infection process of Rhynchosporium secalis in barley using GFP

Through the use of a Rhynchosporium secalis isolate transformed with the green fluorescent protein gene and LASER scanning confocal microscopy (LSCM), fungal development during the R. secalis/barley interaction was analysed. Each infection stage was investigated from 0.5h to 14 days post-inoculation (p.i.) with extensive sampling within the first 48 h p.i. Early germination events were observed that had not been previously described. A specific time of germination was noted, with germ tube formation appearing as early as 1h p.i. Conidia were observed within anticlinal grooves of epidermal cells and the formation of bubbles within these pectin-rich regions was observed within 24h p.i. The study reports R. secalis pectinase production and suggests degradation of these pectin-rich regions. Reactive oxygen species were present during early penetration, 3h p.i. and co-localised with fungal development. LSCM allowed the visualisation of fungal growth deep within tissues at the later stage of the infection.     

Evolution of resistance to Rhynchosporium secalis (Oud.) Davis in barley composite cross II

Summary Changes in resistance to scald disease which occurred in barley composite Cross II over 45 generations were analyzed genetically. This population, which was synthesized in 1929 by pooling equal numbers of f₁ seeds from 378 pair wise crosses among 28 barley varieties, has subsequently been grown at Davis, California under standard agricultural conditions without conscious selection. Progenies derived from self-pollinated seeds from random plants taken from four generations (F₈, F₁₃, F₂₃, and F₄₅) were tested against four different races of scald (40, 61, 72, and 74), and rated as resistant, susceptible or segregating. Striking increases in the frequency of families resistant to races 40, 61, and 74 occurred in CC II. A test for randomness showed that quadruply susceptible and triply resistant families were more common than expected under the assumption that resistance to different races is independent. Positive correlations were found between resistance to races 40, 61, and 74, but resistance to race 72 was independent of resistance to all other races. Possible reasons for these correlations are discussed.     

Molecular mapping of a new gene Rrs4 CI 11549 for resistance to barley scald (Rhynchosporium secalis)

Doubled haploid (DH) progeny from a cross between the scald susceptible barley (Hordeum vulgare L.) cultivar Ingrid and the resistant accession CI 11549 (Nigrinudum) was evaluated for resistance in the pathogen Rhynchosporium secalis (Oudem) J.J. Davis. Two linked and incompletely dominant loci confer resistance CI 11549 against isolate 4004. One is an allele at the complex Rrs1 locus on chromosome 3H close to the centromere; the other is located 22 cM distally on the long arm. The latter locus is designated Rrs4. In BC₃-lines into Ingrid from CI 2222 (another Nigrinudum) resistance seems governed by one locus close to the telomeric region of chromosome 7H, probably allelic to Rrs2. In neither case did we find any trace of the recessive gene rh8 reported to be present in Nigrinudum. Various resistance donors of Ethiopian origin designated as Nigrinudum, Jet or Abyssinian were identical to a great extent with respect to markers, but differed in resistance to different isolates of scald or in barley yellow dwarf virus (BYDV) resistance. The implications for their use as differentials in scald tests and screening of germplasm collections are discussed.     

Differential defense reactions in leaf tissues of barley in response to infection by Rhynchosporium secalis and to treatment with a fungal avirulence gene product

Expression of defense-associated genes was analyzed in leaf tissues of near-isogenic resistant and susceptible barley cultivars upon infection by Rhynchosporium secalis. The genes encoding pathogenesis-related (PR) proteins PR-1, PR-5, and PR-9 are specifically expressed in the mesophyll of resistant plants, whereas a germin-like protein (OxOLP) is synthesized in the epidermis irrespective of the resistance genotype. Restriction-mediated differential display was employed to identify additional epidermis-specific genes. This resulted in the detection of another PR gene, PR-10, along with a lipoxygenase gene, LoxA, and a gene of unknown function, pI2-4, which are specifically induced in the epidermis of resistant plants. The gene encoding a putative protease inhibitor, SD10, is preferentially but not exclusively expressed in the epidermis. The fungal avirulence gene product NIP1 triggers the induction of the four PR genes only. At least two additional elicitors, therefore, must be postulated, one for the unspecific induction of OxOLP and one for the resistance-specific induction of LoxA, pI2-4, and SD10. PR-10 expression can be assumed to be the consequence of NIP1 perception by epidermis cells. In contrast, gene expression in the mesophyll is likely to be triggered by an as yet unknown signal that appears to originate in the epidermis and that is strongly amplified in the mesophyll.     

Isolation of fungal cell wall degrading proteins from barley (Hordeum vulgare L.) leaves infected with Rhynchosporium secalis

Proteins with antifungal activity towards Rhynchosporium secalis conidia were isolated from the intercellular washing fluid (IWF) of barley leaves. The active components were purified by high-performance liquid chromatography under conditions that maintained biological activity. Five major barley IWF proteins deleterious to the cell wall of viable R. secalis conidia were isolated and identified by a combination of N-terminal amino acid sequencing, peptide mapping, and determination of mass and isoelectric point. They were a 32-kDa beta-1,3-glucanase (Pr32), a 25-kDa chitinase (Pr25), and three 22-kDa thaumatin-like (TL) proteins (Pr22-1, Pr22-2, and Pr22-3). Pr22-1 and Pr22-2 were similar to the protein R class of TL proteins, whereas Pr22-3 was more similar to the S class. Pr22-3 was shown to digest laminarin, indicating that this TL protein has glucanase activity. In addition, Pr22-3 was more active in the spore bioassay than Pr22-2. Various combinations of the five proteins had a greater effect on R. secalis spores than did the individual proteins. The extraction of proteins with antifungal activity from the IWF of barley leaves indicates their possible role in defense against leaf pathogens. A similar bioassay may be developed for other systems to identify particular isoforms of pathogenicity-related proteins that might have a role in plant disease resistance.     

Heterologous expression of the avirulence gene product, NIP1, from the barley pathogen Rhynchosporium secalis.

NIP1, the product of the avirulence gene AvrRrs1 from Rhynchosporium secalis, a fungal pathogen of barley, is a small secreted cysteine-rich protein. This protein is essential for the specific recognition of the fungus by host plants carrying the complementary resistance gene Rrs1. Different heterologous expression systems were tested to produce sufficient quantities of NIP1 to allow its utilization in receptor identification and isolation. In addition, protein amounts higher than those produced in fungal cultures are required to determine its 3D structure and to analyze its interaction with a receptor. The most efficient method, the synthesis of a His-tag fusion protein in Escherichia coli combined with a refolding procedure, yielded up to 3 mg of recombinant NIP1 from a 1-liter bacterial culture. After removal of the His-tag, the recombinant protein showed the same physicochemical characteristics as the native NIP1 and, most importantly, full biological activity.     

Resistance to barley scald (Rhynchosporium secalis) in the Ethiopian donor lines 'Steudelli' and 'Jet', analyzed by partial least squares regression and interval mapping

The resistance of barley (Hordeum vulgare L.) to Rhynchosporium secalis (scald) has been investigated in two crosses between the susceptible cv. 'Ingrid' and two resistant Ethiopian landraces, 'Steudelli' and 'Jet'. Doubled haploids were inoculated in replicated tests using two isolates of R. secalis, '4004' and 'WRS1872'. Expression of resistance differed widely between replicated tests. AFLP, SSR and RFLP markers were used to develop chromosome maps. Results have been analysed using partial least squares regression (PLSR) and interval mapping. In PLSR the major covariance structures or 'latent variables' between X (markers) and Y (isolates, tests) are modelled as principal components and their optimal number determined by cross-validation. In 'Steudelli' two QTL were detected, one on each of chromosomes 3H and 7H, in 4 out of 5 tests, while in 'Jet' only one (different) allele at the 3H locus was found. The validated R(2) varied between 11.0% and 64.9% in the replicated tests with '4004'.With isolate 'WRS1872' the 7H locus and another 3H locus were detected. By interval mapping the QTL detected were less stable and generally gave lower R(2) values than PLSR. PLSR does not depend on maps, but interval mapping based on values predicted by PLSR had R(2) around 90%. It is suggested that PLSR may be a useful tool in QTL analysis.     

The effect of nitrogen deficiency on leaf anatomy of young spring barley plants

Spring barley cv. Spartan was cultivated in a complete and nitrogen lacking Richter’s solution. In other experimental variants the nitrogen was omitted after six days of cultivation in the complete nutrient solution or the nitrogen lacking solution was replaced with the complete solution after the same period of time. Anatomy of the second leaf blade was quantitatively analyzed after 12 days of cultivation. Continuous nitrogen deficiency resulted in thinning of the blade, reduction of the cross-section areas of the blade, vascular bundles and sclerenchyma region. The most sensitive reaction to the nitrogen deficiency was that of assimilation parenchyma. The values of characters of the other two investigated variants were between the control and permanent nitrogen lacking variant.     

A single binding site mediates resistance- and disease-associated activities of the effector protein NIP1 from the barley pathogen Rhynchosporium secalis

The effector protein NIP1 from the barley (Hordeum vulgare) pathogen Rhynchosporium secalis specifically induces the synthesis of defense-related proteins in cultivars of barley expressing the complementary resistance gene, Rrs1. In addition, it stimulates the activity of the barley plasma membrane H(+)-ATPase in a genotype-unspecific manner and it induces necrotic lesions in leaf tissues of barley and other cereal plant species. NIP1 variants type I and II, which display quantitative differences in their activities as elicitor and H(+)-ATPase stimulator, and the inactive mutant variants type III* and type IV*, were produced in Escherichia coli. Binding studies using (125)I-NIP1 type I revealed a single class of binding sites with identical binding characteristics in microsomes from near-isogenic resistant (Rrs1) and susceptible (rrs1) barley. Binding was specific, reversible, and saturable, and saturation ligand-binding experiments yielded a K(d) of 5.6 nm. A binding site was also found in rye (Secale cereale) and the nonhost species wheat (Triticum aestivum), oat (Avena sativa), and maize (Zea mays), but not in Arabidopsis (Arabidopsis thaliana). For NIP1 types I and II, equilibrium competition-binding experiments revealed a correlation between the difference in their affinities to the binding site and the differences in their elicitor activity and H(+)-ATPase stimulation, indicating a single target molecule to mediate both activities. In contrast, the inactive proteins type III* and type IV* are both characterized by high affinities similar to type I, suggesting that binding of NIP1 to this target is not sufficient for its activities.     

The race-specific elicitor, NIP1, from the barley pathogen, Rhynchosporium secalis, determines avirulence on host plants of the Rrs1 resistance genotype.

NIP1, a small phytotoxic protein secreted by the barley pathogen Rhynchosporium secalis, is a race-specific elicitor of defense responses in barley cultivars carrying the resistance gene, Rrs1. Co-inoculation employing spores from a virulent fungal race together with the NIP1 protein converted the phenotype of the interaction from compatible to incompatible only on Rrs1-containing plants. In addition, transformation of a virulent fungal race with the nip1 gene yielded avirulent transformants. This demonstrated that the protein is the product of a fungal avirulence gene. The fungal genome was found to contain a single copy of the nip1 gene. Sequence analysis of nip1 cDNA and genomic clones revealed that the gene consists of two exons and one intron. The derived amino acid sequence comprised a secretory signal peptide of 22 amino acids and a cysteine-rich mature protein of 60 amino acids. All fungal races that were avirulent on barley cultivars of the Rrs1 resistance genotype carry and express the nip1 gene and secrete an elicitor-active NIP1 polypeptide. In contrast, races lacking this gene were virulent. In addition, single nucleotide exchanges were detected in the coding region of the nip1 alleles in one virulent fungal race and in a race whose interaction with barley is not controlled by the Rrs1 gene. The resulting exchanges of single amino acids render the gene products elicitor-inactive. Thus, the R.secalis-barley interaction provides the first example of a pathosystem conforming to the gene-for-gene hypothesis in which a plant with a particular resistance gene recognizes a pathogen by a virulence factor, i.e. one of its offensive weapons. On the fungal side, in turn, recognition by the host plant is eluded by either deletion of the encoding gene or alteration of the primary structure of the gene product.