Effect of leaf age and nitrogen fertilisation on sporulation of crown rust (Puccinia coronata var. lolii) on perennial ryegrass (Lolium perenne L.)

Individual leaves of perennial ryegrass cv. Aberystwyth S23 of two leaf ages and at two levels of nitrogen fertilisation were point inoculated with Puccinia coronata f.sp. coronata in a growth chamber. In general, there was no significant difference in the lifespan of inoculated versus control leaves. However, the higher rate of nitrogen extended leaf lifespan more markedly in rusted than in control leaves. Uredospore production varied according to leaf age: colonies on juvenile leaves produced three times as many spores as those on mature leaves.     

Development and characterization of EST-SSR markers for the crown rust pathogen of ryegrass (Puccinia coronata f.sp. lolii).

The causative organism of crown rust in ryegrasses (Puccinia coronata f.sp. lolii) is an obligate biotroph that causes significant economic losses within the temperate grazing industries of dairy, meat, and wool production. This study reports on the development, transferability, and utility of gene-associated simple sequence repeat (SSR) molecular markers for crown rust. Analysis of 1,100 expressed sequence tag (EST) sequences from a urediniospore-derived cDNA library detected 55 SSR loci. The majority of EST-SSR arrays contained perfect trinucleotide repeats with consistently low repeat numbers, and the motifs (ACC)n and (CAT)n were most commonly represented. DNA extraction from single pustules, in conjunction with multiple displacement amplification, provided the basis for PCR-based screening to evaluate genetic marker performance. An example of the identification of intraspecific genetic diversity was obtained from the analysis of 16 P. coronata isolates originating from the United Kingdom, Australia, New Zealand, and Japan. A subset of 12 robust EST-SSR markers was informative for determination of pathogen diversity within and between these localities. It was also demonstrated that crown rust EST-SSR markers were capable of cross-amplification in closely related fungal taxa (Puccinia spp.) and filamentous fungi within the Ascomycota.     

Interaction between crown rust (Puccinia coronata) and two viruses of ryegrass

In Lemtal Italian and S.24 perennial ryegrass plants, two isolates of ryegrass mosaic virus (RMV) suppressed the amount of crown rust emerging on leaves inoculated with Puccinia coronata uredospores by up to 75% compared with the amount on virus-free plants. Severity of rust infection on barley yellow dwarf virus (BYDV) infected plants generally did not differ significantly from that on virus-free plants. When both RMV and BYDV were present, rust was restricted in Lemtal plants to a level intermediate between those occurring on plants infected by either virus alone, and in S.24 plants to a level below that obtained with either virus alone. The mean water soluble carbohydrate (WSC) content of Lemtal plants was reduced more than 20% by RMV, but was not significantly altered by BYDV. In S.24 plants the WSC content was increased by 10% by RMV and by 60% by BYDV. Rust reduced the WSC content of healthy and virus-infected plants, the reduction being positively correlated with the level of rust on the sampled leaves. In plants of Lemtal, but not of S.24, the degree of rust infection was positively correlated with the WSC content of leaves from rust-free control plants.     

Endophyte-mediated disease resistance in wild populations of perennial ryegrass (Lolium perenne)

Twelve wild, endophyte-infected populations of perennial ryegrass were tested for resistance against artificial infection of Drechslera siccans and Fusarium spp. Plants with identified endophyte presence (E+), together with plants free from endophytes (E−), were inoculated with serious turf grass pathogens: D. siccans (cause of brown blight) and a mixture of Fusarium species (cause of Fusarium blight). For both diseases, the positive effect of endophyte presence on plant resistance was observed. In the case of a few ecotypes, endophyte infection increased resistance against both diseases, which is of practical importance for disease control.     

Introgression of crown rust (Puccinia coronata) resistance from meadow fescue (Festuca pratensis) into Italian ryegrass (Lolium multiflorum) and physical mapping of the locus

Resistance was found in the meadow fescue (Festuca pratensis) to crown rust (Puccinia coronata), originating from ryegrasses (Lolium spp). A backcrossing programme successfully transferred this resistance into diploid Italian ryegrass (Lolium multiflorum) and genomic in situ hybridisation (GISH) was used to identify the introgressed fescue chromosome segment. The resistant (R) plants in two BC3 lines all carried an introgressed segment on a single chromosome, which in one of the lines was confined to the short arm of the chromosome. Susceptible (S) plants either contained no introgressed chromosome segment or a segment which was physically smaller than the segments in resistant plants. Using GISH the resistance locus could be physically mapped to the midpoint of a short arm. Segregation ratios of the progeny of BC3 plants, when crossed as R x S and R x R, were in agreement with the hypothesis that the resistance was controlled by a single gene or very closely linked genes. No R plants were produced by crossing S x S plants.     

Genetic control of resistance to crown rust (Puccinia coronato Corda) in Lolium perenne L. and its implications in breeding

Summary A genetic analysis of resistance to crown rust in a series of natural populations of Lolium perenne by means of the diallel cross has revealed that heritability is high (58%) and that the genes concerned show complete but ambi-directional dominance for resistance. An environmental correlation was established between the degree of infection and productivity but no genetic correlation was present. The implications of this mode of control for resistance breeding procedures are briefly considered.     

Development of phosphogluco-isomerase isozymes in perennial ryegrass (Lolium perenne)

The development of isozymes of phosphogluco-isomerase (PGI; D-glucose-6-phosphate ketol isomerase EC 5.3.1.9.) in perennial ryegrasses was followed from dry seed through to leaf senescence using starch gel electrophoretic separations. Root isozymes were also examined. Two forms of the enzyme were found, one (PGI/2) being present in all tissues and at all stages of the life cycle. The other (PGI/1) had two zones of activity, one of which was detected only in light-exposed tissue. Normal development of this form could be inhibited by growing seedlings on distilled water. Some alleles of PGI/2 not previously reported for ryegrasses are also described.     

Acclimation to freezing temperatures in perennial ryegrass (Lolium perenne)

The increasing demands being placed on natural grasslands in the era following the appearance of Bovine Spongiform Encephalitis require that forage crops provide a reliable extended season of growth, combined with good winter survival to ensure sward longevity. The ability to tolerate sub-zero temperatures is integral to the survival of perennial forages. Since the development of freezing tolerance is crucial to the survival and productivity of over-wintering crops, forage breeding programmes require an improved understanding of the individual characteristics that contribute to tolerance to sub-zero temperatures. Photosynthesis, carbohydrate content and changes in protein composition were investigated in two varieties of Lolium perenne which differ in their response to growth at low temperature.     

Identification of QTLs for morphological traits influencing waterlogging tolerance in perennial ryegrass (Lolium perenne L.)

Perennial ryegrass is a globally cultivated obligate outbreeding diploid species (2n = 2x = 14) which is subjected to periods of waterlogging stress due to flood irrigation during winter and the lead-up to summer. Reduction of oxygen supply to root systems due to waterlogging produces consequent deleterious effects on plant performance. Framework genetic maps for a large-scale genetic mapping family [F?(NA(x) × AU?)] were constructed containing 91 simple sequence repeat and 24 single nucleotide polymorphism genetic markers. Genetic trait dissection using both control and waterlogging treatments was performed in the glasshouse, a total of 143 maximally recombinant genotypes being selected from the overall sib-ship and replicated threefold in the trial. Analysis was performed for nine quantitative morphological traits measured 8 weeks after stress treatments were applied. A total of 37 quantitative trait loci (QTLs) were identified; 19 on the NA(x) parental genetic map, and 18 on the AU? parental genetic map. Regions of particular interest were identified on linkage groups (LGs) 4 and 3 of the respective maps, which have been targeted for further analysis by selection of critical recombinants. This first study of genetic control of waterlogging tolerance in ryegrasses has important implications for breeding improvement of abiotic stress adaptation.     

Rds and Rih mediate hypersensitive cell death independent of gene-for-gene resistance to the oat crown rust pathogen Puccinia coronata f. sp. avenae.

The Pca crown rust resistance cluster in the diploid Avena genus confers gene-for-gene specificity to numerous isolates of Puccinia coronata f. sp. avenae. Recombination breakpoint analysis indicates that specificities conferred by the Pca cluster are controlled by at least five distinct genes, designated Pc81, Pc82, Pc83, Pc84, and Pc85. Avena plants with the appropriate genotype frequently respond to P. coronata by undergoing hypersensitive cell death at the sites of fungal infection. Autofluorescence of host cells in response to P. coronata occurs in plants that develop visible necrotic lesions but not in plants that lack this phenotype. Two newly described, non-Pc loci were shown to control hypersensitive cell death. Rds (resistance-dependent suppressor of cell death) suppresses the hypersensitive response (HR), but not the resistance, mediated by the Pc82 resistance gene. In contrast, Rih (resistance-independent hypersensitive cell death) confers HR in both resistant and susceptible plants. Linkage analysis indicates that Rds is unlinked to the Pca cluster, whereas Rih is tightly linked to it. These results indicate that multiple synchronous pathways affect the development of hypersensitive cell death and that HR is not essential for resistance to crown rust. Further characterization of these genes will clarify the relationship between plant disease resistance and localized hypersensitive cell death.     

Physicochemical characterization of cellulose from perennial ryegrass leaves (Lolium perenne)

In this study, we investigated the physicochemical properties of the cellulosic preparations obtained from both untreated perennial ryegrass leaves and de-juiced leaves. It was found that treatment at 22 degrees C with 18% NaOH and 18% KOH for 2h, and 10% NaOH and 10% KOH for 16 h yielded 28.2%, 28.8%, 22.7%, 23.4%, respectively, of 'cellulose' residue from untreated ryegrass leaves and 35.7%, 36.8%, 32.8% and 34.6%, respectively, from the de-juiced leaves. For each cellulosic fraction, the glucose content was 71.6%, 69.6%, 67.8%, 66.7%, 69.7%, 68.6%, 63.9% and 61.7%, respectively. The structure of the cellulose samples was examined using FTIR and CP/MAS (13)C NMR spectroscopy and X-ray diffraction. The cellulosic preparations were free of bound lignin except for noticeable amounts of residual hemicelluloses (28.4-38.3%), and had intrinsic viscosities between 275.1 and 361.0 mL/g, along with molecular weights from 144,130 to 194,930 g/mol. This study found that the cellulose samples isolated from both de-juiced ryegrass leaves and the untreated leaves had a much lower percent crystallinity (33.0-38.6%) than that from wood-based fibres (60-70%) and had much shorter fibres (0.35-0.49 mm) than those of either cereal straws, bagasse or wood. In addition, a partial disruption of the hydrogen bonds and microfibrils may occur during the de-juicing process by mechanical activity, which results in a decreased cellulose crystallinity and fibre length. These findings are significant in relation to hydrolysing ryegrass cellulose for bio-ethanol production.     

Co-transformed, diploid Lolium perenne (perennial ryegrass), Lolium multiflorum (Italian ryegrass) and Lolium temulentum (darnel) plants produced by microprojectile bombardment

A total of 37 plants (30 Lolium multiflorum Lam., 6 L. perenne L., 1 L. temulentum L.) were regenerated from cell suspension colonies bombarded with plasmid DNAs encoding a hygromycin resistance gene (HYG) expressed under a CaMV35S promoter and a β-glucuronidase (GUS) gene expressed under a truncated rice actin1 promoter and first intron, or a maize ubiquitin promoter and first intron. Resistant plants were regenerated under hygromycin selection and transferred to soil. PCR analysis showed that the co-transformation frequency of the GUS gene varied from 33% to 78% of transformants, while histochemical staining of leaf tissue from soil-grown plants showed that the co-expression frequency varied from 37% to 50%. The transgenic nature of the plants was demonstrated by Southern hybridisation analysis, which also showed that the non-selected (GUS) gene was generally present at a higher copy number than the selected (HYG) gene. Received: 10 October 1997 / Revision received: 18 March 1998 / Accepted: 29 November 1998     

Partitioning of sugars in Lolium perenne (perennial ryegrass) during drought and on rewatering

Simulated swards of perennial ryegrass ( Lolium perenne ) growing in 1-m³ soil blocks in the glasshouse were either well watered or deprived of water for 57 d and then rewatered. The first aim was to measure effects of drought on sugar (water-soluble carbohydrate) composition of laminae and sheaths of mature laminae, and bases and laminae of young (growing) leaves. The second aim was to use pulse labelling with ¹⁴CO₂ to follow the partitioning of recently-fixed assimilates, and the assembly and consumption of reserve sugars (fructans). Over the last 7 d of drought growth almost stopped, old leaves died faster than they were replaced, and total sugar (which had doubled in concentration during drought) was rapidly consumed. Leaf laminae had lower content of total sugars and of large fructan (DP>5) than did growing bases and sheaths. Drought greatly reduced the rate at which sugar was exported from the laminae to the sheaths and growing leaf bases, and the rate at which it was converted to fructan. Nevertheless, fructan accumulated over the first 50 d of drought. Rewatering did not result in depolymerization and remobilization of sugars that had been formed during the last 7 d of drought, but stimulated their further assembly into high-DP fructans. Our hypothesis, that accumulation of neo-kestose (a DP-3 fructan) in droughted laminae was a symptom of sugar remobilization just before death, was disproved. It is concluded that sugar reserves contribute to drought resistance only under extreme conditions. The specific role of fructan in dry environments might be to improve regrowth when drought is relieved, rather than to enhance growth during drought.     

Gene-associated single nucleotide polymorphism discovery in perennial ryegrass (Lolium perenne L.)

Molecular genetic marker development in perennial ryegrass has largely been dependent on anonymous sequence variation. The availability of a large-scale EST resource permits the development of functionally-associated genetic markers based on SNP variation in candidate genes. Genic SNP loci and associated haplotypes are suitable for implementation in molecular breeding of outbreeding forage species. Strategies for in vitro SNP discovery through amplicon cloning and sequencing have been designed and implemented. Putative SNPs were identified within and between the parents of the F₁(NA₆ × AU₆) genetic mapping family and were validated among progeny individuals. Proof-of-concept for the process was obtained using the drought tolerance-associated LpASRa2 gene. SNP haplotype structures were determined and correlated with predicted amino acid changes. Gene-length LD was evaluated across diverse germplasm collections. A survey of SNP variation across 100 candidate genes revealed a high frequency of SNP incidence (c. 1 per 54 bp), with similar proportions in exons and introns. A proportion (c. 50%) of the validated genic SNPs were assigned to the F₁(NA₆ × AU₆) genetic map, showing high levels of coincidence with previously mapped RFLP loci. The perennial ryegrass SNP resource will enable genetic map integration, detailed LD studies and selection of superior allele content during varietal development.     

Mapping genes for resistance to barley stripe rust (Puccinia striiformis f. sp. hordei)

Two genes conferring resistance to the barley stripe rust found in Mexico and South America, previously identified as race 24, were mapped to the M arms of barley chromosomes 7 and 4 in a doubled haploid population using molecular markers and the quantitative trait loci (QTL) mapping approach. The resistance gene on chromosome 7 had a major effect, accounting for 57% of the variation in disease severity. The resistance gene on chromosome 4 had a minor effect, accounting for 10% of the variation in trait expression. Two pairs of restriction fragment length polymorphism markers are being used to introgress the resistance genes to North American spring barley using molecular marker-assisted backcrossing.Ore. Agric Exp Stn J no. 10283     

Effect of infection by the endophytic fungus Acremonium lolii on growth and nitrogen uptake by perennial ryegrass (Lolium perenne) in flowing solution culture

Clonal tillers of a genotype of perennial ryegrass (Lolium perenne), either with or without the endophytic fungus Acremonium lolii, were grown under natural light in flowing nutrient solutions with mineral N maintained automatically at concentrations of 3 or 30μm NH₄NO₃ for 28 days. Uptake of N was monitored daily and dry matter production was assessed by sequential harvesting. The presence of endophyte had no significant effect on shoot or root biomass production at either N level, but shoot: root ratios were significantly increased by endophyte infection at both N levels at some harvests. All plants absorbed NH₄⁺ preferentially to NO₃^- and the ratio was not affected by endophyte infection. Also, infection did not affect total N content of plants, which was significantly more in plants at the higher N level than at the lower level. It is concluded that endophyte infection had only minor effects on growth and N economy of the plant, under the conditions imposed in this experiment.