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.     

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.     

Resistance to Rhynchosporium secalis in the winter barley cultivar Vulcan

The six-rowed winter barley cultivar Vulcan was found to be highly resistant to Rhynchosporium secalis in the field, although this resistance was not apparent in routine screening tests of barley genotypes carried out at the seedling stage. More precise seedling tests, using graded inoculum levels to derive dose-response curves, showed that cv. Vulcan had an ED₅₀ which was 4.8 times that of the susceptible, two-rowed cv. Maris Otter, and 4.1 times that of cv. Maris Puma. The inheritance of resistance was investigated at the adult plant stage in a cross with cv. Maris Otter. Although a clear-cut segregation of the F₃ progeny into resistant, segregating and susceptible phenotypes was not observed, the genetic variance of the F₃ family means was compatible with the hypothesis of monogenic control of resistance. Resistance was inherited independently of the factor for six-rowed head type.     

Observations on the production and dispersal of spores, and infection by Rhynchosporium secalis

The numbers of spores of Rhynchosporium secalis washed from samples of barley plants taken weekly, varied markedly. No consistent association with amount of previous rainfall or length of the preceding dry period was detected. On potted seedlings exposed within a crop most infection occurred during long rain periods or when rain fell late in the evening; fewest lesions usually developed on seedlings prevented from touching adjacent plants. Rotorod traps fitted with a 13 mm diameter disc at the apex of each arm were operated under 24 cm diameter covers. Spores were collected on circular cover slips fixed to each disc with glycerine jelly. At all stages of crop growth more spores were trapped at ground level than at other heights tested up to 1 m. The number of spores trapped was not related to the quantity of or duration of rainfall but was related to the mean rate of fall during brief showers only. Efficiency of droplet retention was assessed in a wind tunnel. It declined rapidly when more than c. 5 pl was presented to the disc surface and was less at a wind speed of 1.0 than 0.5 m s-^l. Spore distribution on discs indicated that spores were washed from the surface during long rain periods.     

The inheritance of resistance to Rhynchosporium secalis in some European spring barley cultivars

The inheritance of partial resistance to Rhynchosporium secalis, which appears to be race non-specific, was studied in a diverse range of European spring barley cultivars. Data from the F₂ generation of a 6 × 6 diallel cross and the F₃ generation of three crosses selected from this diallel set suggested that resistance was complex in inheritance, the results being incompatible with any hypothesis involving less than four genes. The F₂ studies indicated that both dominant and recessive genes were active in conferring resistance, and that there were significant additive gene effects. Transgressive segregation occurred in all cross combinations in the F₃ material. Consequently the alleles conferring resistance were not completely concentrated in the most resistant cultivar studied (cv. Proctor). Heritability estimates obtained from F₂ and F₃ material suggested that field selection was of limited reliability, even when spreader drills were incorporated amongst the segregates. Single-plant selection (in F₂) was considered to be of little value, and the results of F_a head-row tests would require confirmation by replicated tests in subsequent generations.     

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.     

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.     

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.     

Relationships between the severity of leaf blotch (Rhynchosporium secalis) and the water-soluble carbohydrate and nitrogen contents of barley plants

Water-soluble carbohydrate (WSC) and total nitrogen (N) contents of leaves of field-grown barley plants were measured in 1966 and 1967. Consistent differences were found between cultivars. The susceptibility of cultivars to leaf blotch was correlated negatively with WSC content and positively with N content at G.S. 10.4. Application of nitrogen fertiliser (which increased N and decreased WSC) also increased the severity of leaf blotch. The occurrence of primary lesions on seedlings does not seem to be affected by their WSC and N contents and the relation between WSC and N contents of leaves and severity of leaf blotch possibly depends on differences in growth and sporulation of the pathogen on substrates containing different amounts of carbohydrate and nitrogen.     

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.     

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.     

Dispersal of Rhynchosporium secalis conidia from infected barley leaves or straw by simulated rain

Simulated rain (mean drop diameter c. 1 or 3 mm) was allowed to fall for 10 – 15 min on to barley leaves or straw infected by Rhynchosporium secalis (leaf blotch). The leaves were supported on a mesh through which run-off water drained and the straw was supported on a rigid surface on which run-off water collected. The numbers of R. secalis conidia and spore-carrying splash droplets collected by horizontal samplers (microscope slides and pieces of photographic film) decreased rapidly with increasing distance from and increasing height above the sources, with half-distances of 2 – 10 cm. Less than 10% of the spores or droplets reached heights of more than 30 cm. Incident drops 3 mm in diameter produced more spore-carrying droplets and dispersed more conidia than did 1 mm drops. The size category of splash droplets with the greatest proportion of the spore-carrying droplets dispersed by 3 mm drops was 200 – 400 μm, whether the source was infected barley leaves or barley straw. For leaves or straw the greatest proportions of spores were carried in droplets > 1000 μm in diameter. The mean diameter of spore-carrying droplets (478 μm) dispersed from free-draining leaves was less than that of droplets from straw plus run-off water (563 μm). However, the leaf source had more spores cm^-2 and the mean number of spores per droplet was greater (113 as opposed to 6·8) than for the straw source.     

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.     

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.     

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.     

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.     

Inheritance of components of partial resistance of barley to Rhynchosporium secalis with particular reference to race specificity

Six spring barley cultivars with no known genes for resistance to specific virulences but varying in partial resistance to Rhynchosporium secalis, were crossed in all combinations (6 × 6 diallel including reciprocals). In addition to seeds from naturally selfed plants, seeds of all parent cultivars were also produced by artificial selfing (emasculation followed by pollination using pollen from the same cultivar). This ensured comparability between seeds of parents and F₁. Both sets of parents, F₁ and F₂ families were grown in the field as single spaced plants and inoculated at Zadoks growth stage 49 with spore suspensions (2 × 10⁶ spores ml^-1) of three races (pathotypes) of R. secalis (Zadoks, Chang & Konzak, 1974). Components of partial resistance, incubation period (ICP), infection frequency (IF) and spore production per lesion (SP/L) were assessed on each plant. There were highly significant differences for all three components of partial resistance in both sets of parent cultivars but rank order in both sets was similar as evidenced by correlation coefficients, r= 0.96 for ICP and IF and r= 0.87 for SP/L. All three components of partial resistance were strongly correlated with NIAB (National Institute of Agricultural Botany, Cambridge, UK) resistance ratings. Means of F₁ and F₂ families were correlated with mid-parent values for ICP and IF but not SP/L. No difference in aggressiveness was found between races but for each component of partial resistance there was a significant interaction between race and parent cultivar (artificial selfs) and, for IF and ICP, a significant interaction between race and F₁ family. There was no evidence of interaction between parent (natural selfs) and race nor between race and F₂ family. Examination of genetic control of resistance showed evidence of strong additive effects (combining ability) in both F₁ and F₂ for ICP and IF but not for SP/L. There was no evidence for maternal or reciprocal differences, but there was evidence for dominance effects although their nature differed between components of partial resistance and between F₁ and F₂ generations. In the F₁, but not the F₂ generation, several elements of dominance (direction, distribution of dominant genes between parent cultivars, specific combining ability) showed for ICP or IF (but not SP/L) significant interaction with race.     

A new method for disease-loss studies used to estimate effects of Pyrenophom teres and Rhynchosporium secalis on winter barley yields

SUMMARY
It may be argued that the basic task of yield-loss studies is to estimate the rate at which a given amount of disease causes a crop to lose yield at each instant during the growing season. This loss rate function can be estimated from detailed data on sets of epidemics and the yields resulting from them in otherwise similar crops. Under certain circumstances, the calculations reduce to regression of yield on integrals of functions of developmental time and disease. Thermal time may form a suitable approximation to developmental time.
The technique was tested using 30 plots 12m × 4 m of winter barley cv. Sonja, in which epidemics of Pyrenophora teres and Rhynchosporium secalis developed. An equation describing loss rate due to P. teres throughout the season and R. secalis in spring was successfully developed. The loss rate due to P. teres was greatest later in the season, and disproportionately severe at low disease levels. On the basis of this experiment the proposed technique compares well with alternatives.