Factors affecting parasitism of stem-borer larvae in Italian ryegrass (Lolium multiflorum)

Factors affecting parasitism of stem-boring larvae which attack Italian ryegrass were studied. Stem-boring larvae dissected out of tillers from a range of Italian ryegrass cultivars showed parasitism of between 10–65%. However, the level of attack by Chasmodon apterus Nees, a ground-dwelling braconid which dominated the parasitoid complex, did appear to be affected by the plant cultivar, independently of the level of stem-borer infestation.Italian ryegrass cultivars, newly-sown on land previously sown to barley and which had received conventional seed-bed cultivations showed low levels of attack by C. apterus, but increased levels of attack by Halticoptera circulus Walker. The use of the pesticide phorate adversely affected parasitism by c. apterus.

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Résumé L'étude porte sur les facteurs influençant le parasitisme par des hyménoptères des larves mineuses des tiges du ray-gras Italien. Le niveau du parasitisme dans les larves extraites des talles de 13 cultivars différents du ray-gras Italien est identique. Toutefois, le niveau d'attaque par Chasmodon apterus, majoritaire dans le complexe des parasites, semble être affecté par le cultivar, quel que soit le niveau d'infestation par les mineuses des tiges. Les effectifs des larves de mineuses des tiges et de C. apterus semblent augmenter parallèlement, le maximum étant atteint avec le cultivar Tiara. Bien que la densité des mineuses soit plus importante pour d'autres cultivars, le niveau de parasitisme y est moindre. Les cultivars de ray-gras Italiens semées après une culture d'orge, et recevant une fertilisation conventionnelle au semis, sont peu attaqués par C. apterus, probablement parce que ce parasite colonise les champs lentement; par contre le niveau d'attaque dû à Halticoptera circulus est accru. Les traitements pesticides à base de phorate affectent négativement le parasitisme par C. apterus.

     

Plant regeneration from ryegrass ovules cultivated on endosperm-derived feeder cells

An efficient method for the regeneration of zygote-derived plants via ovule culture is desirable for overcoming postzygotic cross incompatibility as well as for the development of certain methods for genetic manipulation. High-frequency plantlet regeneration from ovules of Italian ryegrass (Lolium multiflorum Lam.) and a hybrid Italian/perennial ryegrass excised 1 to 4 days post pollination was obtained by culture on endosperm-derived feeder cells. Ovules excised 3 or 4 days after anthesis and grown on feeder cells generally regenerated about twice as frequently as ovules grown directly on nutrient medium. In one of the genotypes tested, ovules excised 1, 2 and 3 days post pollination developed into plantlets at percentages of 38.1, 52.0 and 52.8, respectively, using the feeder-cell system.Abbreviations EM endosperm multiplications - OC ovule culture - R regeneration - 2,4-d 2,4-dichlorophenoxyacetic acid     

Transgenic Italian ryegrass (Lolium multiflorum) plants from microprojectile bombardment of embryogenic suspension cells

Transgenic forage-type Italian ryegrass (Lolium multiflorum Lam.) plants have been obtained by microprojectile bombardment of embryogenic suspension cells using a chimeric hygromycin phosphotransferase (hph) gene construct driven by riceActl 5 regulatory sequences. Parameters for the bombardment of embryogenic suspension cultures with the particle inflow gun were partially optimized using transient expression assays of a chimeric-glucuronidase (gusA) gene driven by the maizeUbi1 promoter. Stably transformed clones were recovered with a selection scheme using hygromycin in liquid medium followed by a plate selection. Plants were regenerated from 33% of the hygromycin-resistant calli. The transgenic nature of the regenerated plants was demonstrated by Southern hybridization analysis. Expression of the transgene in transformed adult Italian ryegrass plants was confirmed by northern analysis and a hygromycin phosphotransferase enzyme assay.Abbreviations 2,4-D 2,4 Dichlorophenoxyacetic acid - GUS Glucuronidase - Hm Hygromycin - HPH Hygromycin phosphotransferase - MS medium Murashige and Skoog medium - PCR Polymerase chain reaction - X-Gluc 5-Bromo-4-chloro--indolyl--D-glucuronic acid     

Genetic mapping reveals a single major QTL for bacterial wilt resistance in Italian ryegrass (Lolium multiflorum Lam.)

Bacterial wilt caused by Xanthomonas translucens pv. graminis (Xtg) is a major disease of economically important forage crops such as ryegrasses and fescues. Targeted breeding based on seedling inoculation has resulted in cultivars with considerable levels of resistance. However, the mechanisms of inheritance of resistance are poorly understood and further breeding progress is difficult to obtain. This study aimed to assess the relevance of the seedling screening in the glasshouse for adult plant resistance in the field and to investigate genetic control of resistance to bacterial wilt in Italian ryegrass (Lolium multiflorum Lam.). A mapping population consisting of 306 F₁ individuals was established and resistance to bacterial wilt was assessed in glasshouse and field experiments. Highly correlated data (r = 0.67–0.77, P < 0.01) between trial locations demonstrated the suitability of glasshouse screens for phenotypic selection. Analysis of quantitative trait loci (QTL) based on a high density genetic linkage map consisting of 368 amplified fragment length polymorphism (AFLP) and simple sequence repeat (SSR) markers revealed a single major QTL on linkage group (LG) 4 explaining 67% of the total phenotypic variance (Vp). In addition, a minor QTL was observed on LG 5. Field experiments confirmed the major QTL on LG 4 to explain 43% (in 2004) to 84% (in 2005) of Vp and also revealed additional minor QTLs on LG 1, LG 4 and LG 6. The identified QTLs and the closely linked markers represent important targets for marker-assisted selection of Italian ryegrass.     

The effects of low,regulated supplies of nitrate and ammonium nitrogen on the growth and composition of perennial ryegrass

Summary Perennial ryegrass was grown in flowing solution culture with nitrogen supplied in amounts that increased exponentially,i.e. in parallel with the rate of increase in growth. Nitrogen was supplied as either NO ₃ ⁻ or NH ₄ ⁺ , and the amounts to be added were calculated on the basis of extrapolated values for dry weights obtained from fitted curves. There were two rates of addition for each form of N aimed at providing adequate (5.0 per cent) and less than adequate (2.75 per cent) contents in the plants in each case. Measured plant weights and N concentrations were in close agreement with predicted values over a four week experimental period. There was no effect of N-form at high N, and these plants produced 46 per cent more dry matter than the plants at low N. Only minor differences in overall growth occurred with NO ₃ ⁻ or NH ₄ ⁺ plants at low N, but the NH ₄ ⁺ plants had a greater shoot:root ratio. The absorption rate (m mol Ng⁻ root d⁻¹) for NH ₄ ⁺ -N was therefore greater than for NO ₃ ⁻ -N. The cation/anion composition of the plants was affected in a predicable way, and to a greater or lesser extent at high or low N, respectively, in NO ₃ ⁻ or NH ₄ ⁺ plants. The major changes in cation composition came through effects on potassium absorption. Plants with low NO ₃ ⁻ appeared to be under greater N stress than those with low NH ₄ ⁺ because of the lower shoot:root ratio and the greater C∶N ratio in the shoots.     

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.     

Osmoregulation and role of nitrate during regrowth after cutting of ryegrass (Lolium perenne)

The osmotic role of nitrate during aftermath growth of Lolium perenne L. cv. Réveille was investigated. Plants were grown from seed in a controlled environment using a liquid medium with 1.0 m M NH₄NO₃ as nitrogen source.
Eight-week-old plants were cut 4.0 cm above the root system and then harvested over a 14-day period of regrowth on the same initial nutrient solution, except that nitrate was ¹⁵N labelled. Throughout the experimental period, nitrate storage and reduction in roots were low. In stubble and especially in leaves, nitrate accumulated during the first 6 days of regrowth whereas nitrate reduction mainly occurred after this period. Analyses of carbohydrate, chloride and potassium contents in stubble and leaves showed that the accumulation of nitrate osmotically compensated for the decrease in soluble sugars during the first 6 days of regrowth.
The cumulative osmotic potential of sugars, chloride and nitrate in differently treated plants was studied in stubble and leaves. Compared with uncut plants, the lower carbohydrate concentrations found in cut plants regrowing on 1.0 m M NH₄NO₃ were compensated for by an accumulation of nitrate. During aftermath growth on low nitrogen nutrition (0.2 m M NH₄NO₃), chloride replaced nitrate, supporting the proposed osmotic function of nitrate.
It is concluded that nitrate is involved in the osmotic adjustment of ryegrass during regrowth after cutting.     

Asymmetric somatic hybridization between tall fescue (Festuca arundinacea Schreb.) and irradiated Italian ryegrass (Lolium multiflorum Lam.) protoplasts

Intergeneric asymmetric somatic hybrids have been obtained by the fusion of metabolically inactivated protoplasts from embryogenic suspension cultures ofFestuca arundinacea (recipient) and protoplasts from a non-morphogenic cell suspension ofLolium multiflorum (donor) irradiated with 10, 25, 50, 100, 250 and 500 Gy of X-rays. Regenerating calli led to the recovery of genotypically and phenotypically different asymmetric somatic hybridFestulolium plants. The genome composition of the asymmetric somatic hybrid clones was characterized by quantitative dot-blot hybridizations using dispersed repetitive DNA sequences specific to tall fescue and Italian ryegrass. Data from dot-blot hybridizations using two cloned Italian ryegrass-specific sequences as probes showed that irradiation favoured a unidirectional elimination of most or part of the donor chromosomes in asymmetric somatic hybrid clones obtained from fusion experiments using donor protoplasts irradiated at doses 250 Gy. Irradiation of cells of the donor parent with 500 Gy prior to protoplast fusion produced highly asymmetric nuclear hybrids with over 80% elimination of the donor genome as well as clones showing a complete loss of donor chromosomes. Further information on the degree of asymmetry in regenerated hybrid plants was obtained from chromosomal analysis including in situ hybridizations withL. multiflorum-specific repetitive sequences. A Southern blot hybridization analysis using one chloroplast and six mitochondrial-specific probes revealed preferentially recipient-type organelles in asymmetric somatic hybrid clones obtained from fusion experiments with donor protoplasts irradiated with doses higher than 100 Gy. It is concluded that the irradiation of donor cells before fusion at different doses can be used for producing both nuclear hybrids with limited donor DNA elimination or highly asymmetric nuclear hybrid plants in an intergeneric graminaceous combination. For a wide range of radiation doses tested (25–250Gy), the degree of the species-specific genome elimination from the irradiated partner seems not to be dose dependent. A bias towards recipient-type organelles was apparent when extensive donor nuclear genome elimination occurred.Abbreviations cpDNA Chloroplast DNA - 2, 4-D 2,4-dichlorophenoxyacetic acid - FDA fluorescein diacetate - IOA iodoacetamide - mtDNA mitochondrial DNA - RFLP restriction fragment length polymorphism     

Intergeneric somatic hybridization in Gramineae: somatic hybrid plants between tall fescue (Festuca arundinacea Schreb.) and Italian ryegrass (Lolium multiflorum Lam.)

Summary Tall fescue (Festuca arundinacea Schreb.) protoplasts, inactivated by iodoacetamide, and non-morphogenic Italian ryegrass (Lolium multiflorum Lam.) protoplasts, both derived from suspension cultures, were electrofused and putative somatic hybrid plants were recovered. Two different genotypic fusion combinations were carried out and several green plants were regenerated in one of them. With respect to plant habitus, leaf and inflorescence morphology, the regenerants had phenotypes intermediate between those of the parents. Southern hybridization analysis using a rice ribosomal DNA probe revealed that the regenerants contained both tall fescue- and Italian ryegrass-specific-DNA fragments. A cloned Italian ryegrass-specific interspersed DNA probe hybridized to total genomic DNA from Italian ryegrass and from the green regenerated somatic hybrid plants but not to tall fescue. Chromosome counts and zymograms of leaf esterases suggested nuclear genome instability of the somatic hybrid plants analyzed. Four mitochondrial probes and one chloroplast DNA probe were used in Southern hybridization experiments to analyze the organellar composition of the somatic hybrids obtained. The somatic hybrid plants analyzed showed tall fescue, additive or novel mtDNA patterns when hybridized with different mitochondrial gene-specific probes, while corresponding analysis using a chloroplast gene-specific probe revealed in all cases the tall fescue hybridization profile. Independently regenerated F. arundinacea (+) L. multiflorum somatic hybrid plants were successfully transferred to soil and grown to maturity, representing the first flowering intergeneric somatic hybrids recovered in Gramineae.     

Water use and chemical composition of ryegrass (Lolium) cultivars

Summary Significant differences in total dry matter yields of shoots and roots were found between 11 ryegrass (Lolium) cultivars grown in a glasshouse. Although shoot yield varied significantly between individual cultivars there was no overall difference between the annual and perennial cultivars; whereas for roots, the yields of the perennial plants were much smaller than those of the annual types. Water use (g H₂O g total DM^–1) also varied significantly between cultivars. However, there was no relationship between efficient water use and dry matter production.No significant differences were found in shoot composition between the cultviars for nitrogen, phosphorus, and potassium; however, concentrations of sulphur, magnesium, calcium, and sodium varied significantly. Sodium concentrations were generally higher in the annual compared to the perennial cultivars. For roots only nitrogen, phosphorus, and sulphur differed significantly between cultivars. Of the elements only calcium in the shoots was shown to be related to water use. Thus cultivars which were low users of water also had significantly lower calcium concentrations in their shoots. Water use appeared to affect the absorption of calcium by the root to a far greater extent than the transport from roots to shoot. An apparent relationship between magnesium concentration in the shoots and water use was shown to be due to the close association of magnesium with calcium in the plant.     

The effect of nitrate concentration in a flowing solution system on growth and nitrate uptake of twoPlantago species

Summary Juvenile plants ofPlantago lanceolata andP. major ssp.major were grown in a flowing solution system at 7.5 mM or 9.5 M NO₃. The parameters investigated were: RGR, shoot weight percentage, leaf length, length of main root axis, shoot concentrations of major ions and organic N, and the specific uptake rate for NO₃. At 9.5 M NO₃ growth ofP. major was not hampered, whereas shoot growth and leaf length ofP. lanceolata were reduced. The NO₃ concentration ofP. lanceolata decreased more than that ofP. major. The different performances of the species at 9.5 M NO₃ were associated with different specific uptake rates. In both treatments the root system ofP. major was shorter than that ofP. lanceolata. P. lanceolata accumulated more NO₃ in the leaves. The performance of thePlantago species is discussed in relation to the availability of nutrients in their habitats.Grassland Species Research Group. Publication no. 37.     

Nitrogen transfer from arrowleaf clover to ryegrass in field plantings

Arrowleaf clover (Trifolium vesiculosum Savi) and annual ryegrass Lolium multiflorum Lam.) commonly are overseeded in dormant bermudagrass (Cynodon dactylon L. Pers.) sod on coastal plain soils in the southeastern United States. Two field experiments were conducted in consecutive years at different sites to estimate the amount of N transferred from the clover to the annual grass. Nitrogen treatments included 50 kg N ha^-1 as ¹⁵N depleted ammonium nitrate applied in either February or April, and a check (no N applied). Three clippings were made during the cool-season from March to June. In both experiments, less than 5 kg N ha^-1 were transferred from the clover to the grass. Ryegrass yields of dry matter and total N were not increased by growing with clover. Clover growth was typical for the region; average dry matter yield in pure stand was 2,615 kg ha^-1 over the two-year period. Clover in mixed stand fixed between 20 and 60 kg N/ha. Less than 13% of N contained in ryegrass was transferred from arrowleaf clover to ryegrass at any clipping while clover was actively growing. The quantity of N transferred over the entire season was not statistically significant.     

Net Mg2+ uptake in relation to the amount of exchangeable Mg2+ in the Donnan free space of ryegrass roots

Ammonium acetate and BaCl₂-triethanolamine were used to desorb Mg²⁺ from the root Donnan free space (DFS) of 23-d-old ryegrass (Lolium multiflorum Lam. cvs. Gulf and Wilo). Amounts of desorbed Mg²⁺ increased with the increase in Mg²⁺ activity of the nutrient solution. Slightly less Mg²⁺ was desorbed by Ba²⁺ than by NH₄ ⁺. Previously published data on short-term net Mg²⁺ uptake by intact 23-d-old ryegrass plants of the two cultivars were linearly related to the amount of exchangeable Mg⁺ desorbed from the root DFS (r²=0.90 and 0.81 for the desorption by NH₄ ⁺ and Ba²⁺, respectively). A sward of Mg²⁺ ions attracted to the negative charges of the cell surface is suggested to represent a part of a pool of Mg²⁺ available for active transport through the plasmalemma.     

The roles of nitrate and ammonium in the regulation of the development of nitrate reductase in Chlamydomonas reinhardii

The regulation of the development of nitrate reductase (NR) activity in Chlamydomonas reinhardii has been compared in a wild-type strain and in a mutant (nit-A) which possesses a modified nitrate reductase enzyme that is non-functional in vivo. The modified enzyme cannot use NAD(P)H as an electron donor for nitrate reduction and it differs from wild-type enzyme in that NR activity is not inactivated in vitro by incubation with NAD(P)H and small quantities of cyanide; it is inactivated when reduced benzyl viologen or flavin mononucleotide is present. After short periods of nitrogen starvation mutant organisms contain much higher levels of terminal-NR activity than do similarly treated wild-type ones. Despite the inability of the mutant to utilize nitrate, no nitrate or nitrite was found in nitrogen-starved cultures; it is therefore concluded that the appearance of NR activity is not a consequence of nitrification. After prolonged nitrogen starvation (22 h) the NR level in the mutant is low. It increases rapidly if nitrate is then added and this increase in activity does not occur in the presence of ammonium, tungstate or cycloheximide. Disappearance of preformed NR activity is stimulated by addition of tungstate and even more by addition of ammonium. The results are interpreted as evidence for a continuous turnover of NR in cells of the mutant with ammonium both stimulating NR breakdown and stopping NR synthesis. Nitrate protects the enzyme from breakdown. Reversible inactivation of NR activity is thought to play an insignificant rôle in the mutant.Abbreviations NR nitrate reductase - BV benzyl viologen     

Aluminum and plant age effects on adsorption of cations in the Donnan free space of ryegrass roots

Four cultivars of ryegrass (Lolium multiflorum Lam. cvs. Gulf, Marshall, Urbana, and Wilo) were grown in nutrient solution (pH 4.2) at two Al levels (0 and 74 μM). Cations were desorbed from the Donnan free space of roots of 15-, 23-, and 35-day-old plants using BaCl₂, BaCl₂-triethanolamine, NH₄OAc, and KCl. The amounts of desorbed Ca²⁺ and K⁺ decreased, while desorption of Mn²⁺ and Na⁺ increased with plant age. Differences between 15- and 35-day-old plants, but not between 15- and 23-day-old, were significant. Aluminum considerably decreased the amount of desorbed divalent cations (Ca²⁺, Mg²⁺) and increased the amount of desorbed K⁺ and Na⁺. Ability to resist these changes appeared to be one of the mechanisms determining Al tolerance of ryegrass cultivars.     

Control and variation ofin vivo nitrate reductase activity inLolium perenne L. cv S24

Summary Variation in nitrate reductase activity associated with vegetative development and morphological differences inLolium perenne L. cv S24 was examined using anin vivo technique of enzyme assay. Little difference in enzyme activity per unit weight of leaf laminar tissue was found between tillers, between and within leaf laminae but activity in leaf laminae, was much higher than that of leaf sheaths; the activity of shoot material was greater than the apparent root activity. Senescence of leaf material was not accompanied by marked changes invivo nitrate reductase activity.     

Consistent detection of QTLs for crown rust resistance in Italian ryegrass (<Emphasis Type="Italic">Lolium multiflorum</Emphasis> Lam.) across environments and phenotyping methods

Crown rust, caused by Puccinia coronata f. sp. lolii, is one of the most important diseases of temperate forage grasses, such as ryegrasses (Lolium spp.), affecting yield and nutritional quality. Therefore, resistance to crown rust is a major goal in ryegrass breeding programmes. In a two-way pseudo-testcross population consisting of 306 Lolium multiflorum individuals, multisite field evaluations as well as alternative methods based on artificial inoculation with natural inoculate in controlled environments were used to identify QTLs controlling resistance to crown rust. Disease scores obtained from glasshouse and leaf segment test (LST) evaluations were highly correlated with scores from a multisite field assessment (r = 0.66 and 0.79, P < 0.01, respectively) and thus confirmed suitability of these methods for crown rust investigations. Moreover, QTL mapping based on a linkage map consisting of 368 amplified fragment length polymorphism (AFLP) and simple sequence repeat (SSR) markers revealed similar results across different phenotyping methods. Two major QTLs were consistently detected on linkage group (LG) 1 and LG 2, explaining up to 56% of total phenotypic variance (V _p). Nevertheless, differences between position and magnitude of QTLs were observed among individual field locations and suggested the existence of specific local pathogen populations. The present study not only compared QTL results among crown rust evaluation methods and environments, but also identified molecular markers closely linked to previously undescribed QTLs for crown rust resistance in Italian ryegrass with the potential to be applied in marker-assisted forage crop breeding. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

A comparison of the high-active and low-active form of nitrate reductase in synchronous Chlorella sorokiniana

Chlorella sorokiniana possesses two forms of nitrate reductase (EC 1.6.6.1.). One with low activity is present in cells at the end of the light-dark cycle, the other with high activity is present after 1 h of illumination. The two forms can be distinguished by gel electrophoresis, isopycnic centrifugation, assay of the partial reactions and their sensitivity to antibodies, respectively. These differences are discussed with respect to an effect of intracellular nitrate on the activation of nitrate reductase.Abbreviations NAR nitrate reductase - FMN flavine mononucleotide - MV methylviologen     

Relationships between external nitrate availability,nitrate uptake and expression of nitrate reductase in roots of barley grown in N-limited split-root cultures

Despite the large number of studies of nitrate metabolism in plants, it remains undetermined to what extent this key plant system is controlled by overall plant N nutrition on the one hand, and by the nitrate ion itself on the other hand. To investigate these questions, V _max for nitrate uptake (high-affinity range), and nitrate reductase (NR) mRNA and activity, were measured in roots of N-limited barley (Hordeum vulgare L. cv. Golf) grown under conditions of constant relative addition of nitrate, with the seminal roots split between two culture compartments. The total amount of nitrate added per unit time (0.09·d^-1) was distributed between the two root parts (subroots) in ratios of 1000, 982, 955, 9010, 8020, and 5050. These nitrate-addition ratios resulted in nitrate fluxes ranging from 0 to 23 mol nitrate·g^-1 DW root·h^-1, while the external nitrate concentrations varied between 0 and 1.2 M. The apparent V _max for net nitrate uptake showed saturation-type responses to nitrate flux maintained during preceding growth. The flux resulting in half-maximal induction of nitrate uptake was approximately 4 mol nitrate·g^-1 DW root·h^-1, corresponding to an external nitrate concentration of 0.7 M. The activity of NR and levels of NR mRNA did not saturate within the range of nitrate fluxes studied. None of the parameters studied saturated with respect to the steady-state external nitrate concentration. At the zero nitrate addition — the 0%-root — initial uptake activity as determined in short-term ¹⁵N-labelling experiments was insignificant, and NR activity and NR mRNA were not detectable. However, nitrate uptake was rapidly induced, showing that the 0%-root had retained the capacity to respond to nitrate. These results suggest that local nitrate availability has a significant impact on the nitrate uptake and reducing systems of a split-root part when the total plant nitrate nutrition is held constant and limiting.Abbreviation NR nitrate reductase This work was supported by the Lars Hierta Memory Foundation, the Royal Swedish Academy of Sciences, and by the Swedish Natural Science Research Council via project grants (to C.-M.L. and B.I.) and visiting scientist grant (to W.H.C.). We thank Mrs. Ellen Campbell for technical advice, and Mrs. Judith V. Purves, Long Ashton Research Station, Long Ashton, UK, for analyses of ¹⁵N-labelling in tissue samples.