Functionally associated molecular genetic marker map construction in perennial ryegrass (Lolium perenne L.)

A molecular marker-based map of perennial ryegrass (Lolium perenne L.) has been constructed through the use of polymorphisms associated with expressed sequence tags (ESTs). A pair-cross between genotypes from a North African ecotype and the cultivar Aurora was used to generate a two-way pseudo-testcross population. A selection of 157 cDNAs assigned to eight different functional categories associated with agronomically important biological processes was used to detect polymorphic EST–RFLP loci in the F1(NA6 × AU6) population. A comprehensive set of EST–SSR markers was developed from the analysis of 14,767 unigenes, with 310 primer pairs showing efficient amplification and detecting 113 polymorphic loci. Two parental genetic maps were produced: the NA6 genetic map contains 88 EST–RFLP and 71 EST–SSR loci with a total map length of 963 cM, while the AU6 genetic map contains 67 EST–RFLP and 58 EST–SSR loci with a total map length of 757 cM. Bridging loci permitted the alignment of homologous chromosomes between the parental maps, and a sub-set of genomic DNA-derived SSRs was used to relate linkage groups to the perennial ryegrass reference map. Regions of segregation distortion were identified, in some instances in common with other perennial ryegrass maps. The EST-derived marker-based map provides the basis for in silico comparative genetic mapping, as well as the evaluation of co-location between QTLs and functionally associated genetic loci.

Response of perennial ryegrass (Lolium perenne) to renovation in Australian dairy pastures

This study reports on the effect of oversowing perennial ryegrass (Lolium perenne L.) into a degraded perennial ryegrass and white clover (Trifolium repens L.) pasture to extend its productive life using various intensities of seedbed preparation. Sites in New South Wales (NSW), Western Australia (WA), South Australia (SA) and Tasmania (Tas.) were chosen by a local group of farmers as being degraded and in need of renovation. Control (nil renovation) and medium (mulch and graze, spray with glyphosphate and sow) renovation treatments were common to all sites whereas minimum (mulch and graze, and sow) and full seedbed (graze and spray with glyphosphate and then full seedbed preparation) renovation were imposed only at some sites. Plots varied in area from 0.14 to 0.50 ha, and were renovated then sown in March or April 2000 and subsequently grazed by dairy cows. Pasture utilisation was estimated from pre- and post-grazing pasture mass assessed by a rising plate pasture meter. Utilised herbage mass of the renovated treatments was significantly higher than control plots in period 1 (planting to August) and 2 (first spring) at the NSW site only. There was no difference among treatments in period 3 (first summer) at any site, and only at the WA and NSW sites in period 4 (March to July 2001) was there a response to renovation. As a result, renovation at the NSW site only significantly increased ryegrass utilisation over the whole experimental period. Ryegrass plant density was higher at the NSW, WA (excluding minimum renovation) and Tas. (excluding full renovation) sites 6 months after renovation but this was only sustained for 12 months for the minimum and medium treatments at the NSW and Tas. sites, respectively, presumably due to reduced competition from naturalised C4 summer grasses [kikuyu (Pennisetum clandestinum) and paspalum (Paspalum dilatatum)] in NSW At the NSW, WA and SA sites, the original ryegrass plant density was low (<35 plants/m2) compared with the Tas. site where density was around 185/m2. The response to renovating a degraded perennial ryegrass pasture varied between sites in Australia. Positive responses were generally small and were most consistent where renovation removed competing C4 summer grasses.

Comparative productivity of irrigated short-term ryegrass (Lolium multiflorum) pasture receiving nitrogen, grown alone or in a mixture with white (Trifolium repens) and Persian (T. resupinatum) clovers

Dairy farms in subtropical Australia use irrigated, annually sown short-term ryegrass (Lolium multiflorum) or mixtures of short-term ryegrass and white (Trifolium repens) and Persian (shaftal) (T. resupinatum) clover during the winter-spring period in all-year-round milk production systems. A series of small plot cutting experiments was conducted in 3 dairying regions (tropical upland, north Queensland, and subtropical southeast Queensland and northern New South Wales) to determine the most effective rate and frequency of application of nitrogen (N) fertiliser. The experiments were not grazed, nor was harvested material returned to the plots, after sampling. Rates up to 100 kg N/ha.month (as urea or calcium ammonium nitrate) and up to 200 kg N/ha every 2 months (as urea) were applied to pure stands of ryegrass in 1991. In 1993 and 1994, urea, at rates up to 150 kg N/ha.month and to 200 kg N/ha every 2 months, was applied to pure stands of ryegrass; urea, at rates up to 50 kg N/ha.month, was also applied to ryegrass-clover mixtures. The results indicate that applications of 50-85 kg N/ha.month can be recommended for short-term ryegrass pastures throughout the subtropics and tropical uplands of eastern Australia, irrespective of soil type. At this rate, dry matter yields will reach about 90% of their potential, forage nitrogen concentration will be increased, there is minimal risk to stock from nitrate poisoning and there will be no substantial increase in soil N. The rate of N for ryegrass-clover pastures is slightly higher than for pure ryegrass but, at these rates, the clover component will be suppressed. However, increased ryegrass yields and higher forage nitrogen concentrations will compensate for the reduced clover component. At application rates up to 100 kg N/ha.month, build-up of NO3--N and NH4+-N in soil was generally restricted to the surface layers (0-20 cm) of the soil, but there was a substantial increase throughout the soil profile at 150 kg N/ha.month. The build-up of NO3--N and NH4+-N was greater and was found at lower rates on the lighter soil compared with heavy clays. Generally, most of the soil N was in the NO3--N form and most was in the top 20 cm.

The effect of endophyte on the performance of irrigated perennial ryegrasses in subtropical Australia

The effect of fungal endophyte (Neotyphodium lolii) infection on the performance of perennial ryegrass (Lolium perenne) growing under irrigation in a subtropical environment was investigated. Seed of 4 cultivars, infected with standard (common toxic or wild-type) endophyte or the novel endophyte AR1, or free of endophyte (Nil), was sown in pure swards, which were fertilised with 50 kg N/ha.month. Seasonal and total yield, persistence, and rust susceptibility were assessed over 3 years, along with details of the presence of endophyte and alkaloids in plant shoots. Endophyte occurrence in tillers in both the standard and AR1 treatments was above 95% for Bronsyn and Impact throughout and rose to that level in Samson by the end of the second year. Meridian AR1 only reached 93% while, in the standard treatment, the endophyte had mostly died before sowing. Nil Zendophyte treatments carried an average of ?0.6% infection throughout. Infection of the standard endophyte was associated with increased dry matter (DM) yields in all 3 years compared with no endophyte. AR1 also significantly increased yields in the second and third years. Over the full 3 years, standard and AR1 increased yields by 18% and 11%, respectively. Infection with both endophytes was associated with increased yields in all 4 seasons, the effects increasing in intensity over time. There was 27% better persistence in standard infected plants compared with Nil at the end of the first year, increasing to 198% by the end of the experiment, while for AR1 the improvements were 20 and 134%, respectively. The effect of endophyte on crown rust (Puccinia coronata) infection was inconsistent, with endophyte increasing rust damage on one occasion and reducing it on another. Cultivar differences in rust infection were greater than endophyte effects. Plants infected with the AR1 endophyte had no detectable ergovaline or lolitrem B in leaf, pseudostem, or dead tissue. In standard infected plants, ergovaline and lolitrem B were highest in pseudostem and considerably lower in leaf. Dead tissue had very low or no detectable ergovaline but high lolitrem B concentrations. Peramine concentration was high and at similar levels in leaf and pseudostem, but not detectable in dead material. Concentration was similar in both AR1 and standard infected plants. Endophyte presence appeared to have a similar effect in the subtropics as has been demonstrated in temperate areas, in terms of improving yields and persistence and increasing tolerance of plants to stress factors.

Evaluating temperate species for the subtropics. 1. Annual ryegrasses

In the subtropics of Australia, irrigated temperate species are the key to reliable cool season feed on dairy farms. Persistence of perennial species is a major limitation to achieving reliable production from irrigated areas and yearly sowings of annual ryegrasses have replaced them as the most productive cool season forage production system in the subtropics. This series of experiments evaluated the yield, and resistance to rust damage, of commercially available cultivars and breeders' lines of annually sown ryegrasses (Lolium multiflorum, L. rigidum, L. x boucheanum and L perenne) in pure, nitrogen-fertilised swards under irrigation in the subtropics over a 22-year period. Barberia and Aristocrat 2 were the most adapted cultivars for subtropical conditions, producing high yields (119 and 114% of mean yield, respectively) and demonstrating the least rust damage. Newer selections from New Zealand, South African, United States of America and European breeding programs are performing better under subtropical conditions than older cultivars, particularly if a component of the selection process has been conducted in that environment. Cultivars such as Passerei Plus, Crusader, Hulk, Status and Warrior are examples of this process, producing between 105 and 115% of mean yield. Yields of annual ryegrass cultivars, which have been available or still are available for sale in Australia, ranged from 14-30 t/ha DM, depending on cultivar, site and seasonal conditions. Yields were lower at the site, which had inferior soil structure and drainage. Up to 50% of yield was produced in the 3 winter months. There was a trend towards improved yields and better tolerance of crown rust from experimental lines in the subtropics, as breeders strive for wider adaptation. Around 70% of the variation in total yield of annual ryegrass and 50 and 60% of the variation in winter and spring yield, respectively, were significantly explained by cultivar, site and climatic variables in autumn, winter and spring. While level of rust damage had no effect on total or seasonal yields, it affected the amount of green leaf available in spring. Under subtropical conditions, winter, spring and overall (autumn to mid-summer) temperatures influenced the- development of rust, which along with cultivar, accounted for 46% of the variation in rust damage. Cultivars showed a range of adaptation, with some performing well only under adverse conditions, some being well adapted to all conditions and some which performed well only under favoured conditions. Cultivars with high winter yields were most suited to subtropical conditions and included Aristocrat 2 (now released as CM 108), Barberia, Warrior, Crusader, Status, Passerei Plus and Hulk. Short growing season types such as Winter Star and T Rex performed well in winter but achieved lower total production, and long season cultivars such as Flanker rarely achieved their potential because of unfavourable conditions in late summer.

Ruminal protein degradability of a range of tropical pastures

The rumen degradability parameters of the diet selected by two to four oesophageal-fistulated Brahman steers grazing a range of tropical pastures were determined by incubation of extrusa in nylon bags suspended in the rumen of rumen-fistulated (RF) Brahman steers. The effective protein degradability (Edg) was determined by measuring the rate of disappearance of neutral detergent insoluble nitrogen (NDIN) less acid detergent insoluble nitrogen (ADIN) in the incubated extrusa. Six to eight RF steers also grazed each of the pastures along with the oesophageal-fistulated steers, to allow determination of key rumen parameters and rumen particulate matter fractional outflow rates (FOR). The seven pastures studied included: native tropical grass (C4) pasture (major species Heteropogon contortus and Bothriochloa bladhii), studied in the early wet (NPEW), the wet/dry transition (NPT) and the dry (NPD) seasons; introduced tropical grass (C4) pasture (Bothriochloa insculpta), studied in the mid wet season (BB); the introduced tropical legumes (C3), Lablab purpureus (LL) and Clitoria ternatea (BP); and the temperate grass (C3) pasture, ryegrass (Lolium multiflorum, RG). Using the measured particle FOR values in calculations, the Edg estimates were very high for both C4 and C3 species: 0.82–0.91 and 0.95–0.98 g/g crude protein (CP), respectively. Substitution of an assumed FOR (kp = 0.02/h) for the measured values for each pasture type did not markedly affect estimates of Edg. However, C4 tropical grasses had much lower effective rumen degradable protein (ERDP) fractions (23–66 g/kg DM) than the C3 pasture species RG and LL (356 and 243 g/kg DM, respectively). This was associated with a lower potential degradability and degradation rate of organic matter (OM) in sacco, lower in vitro organic matter digestibility (IVOMD) and CP concentrations in the extrusa, and lower ammonia-N and branched-chain fatty acid concentrations in rumen fluid for the tropical grasses. As tropical grass pastures senesced, there was a decline in Edg, the ERDP and rumen undegradable protein (UDP) fractions, the potential degradability and degradation rate of OM and the IVOMD. These results provide useful data for estimating protein supply to cattle grazing tropical pastures.

The performance of irrigated mixtures of tall fescue, ryegrass and white clover in subtropical Australia. 2. The effects on yield and botanical composition of managing for quality.

The effects on yield, botanical composition and persistence, of using a variable defoliation schedule as a means of optimising the quality of the tall fescue component of simple and complex temperate pasture mixtures in a subtropical environment was studied in a small plot cutting experiment at Gatton Research Station in south-east Queensland. A management schedule of 2-, 3- and 4-weekly defoliations in summer, autumn and spring and winter, respectively, was imposed on 5 temperate pasture mixtures: 2 simple mixtures including tall fescue (Festuca arundinacea) and white clover (Trifolium repens); 2 mixtures including perennial ryegrass (Lolium perenne), tall fescue and white clover; and a complex mixture, which included perennial ryegrass, tall fescue, white, red (T. pratense) and Persian (T. resupinatum) clovers and chicory (Cichorium intybus). Yield from the variable cutting schedule was 9% less than with a standard 4-weekly defoliation. This loss resulted from reductions in both the clover component (13%) and cumulative grass yield (6%). There was no interaction between cutting schedule and sowing mixture, with simple and complex sowing mixtures reacting in a similar manner to both cutting schedules. The experiment also demonstrated that, in complex mixtures, the cutting schedules used failed to give balanced production from all sown components. This was especially true of the grass and white clover components of the complex mixture, as chicory and Persian clover components dominated the mixtures, particularly in the first year. Quality measurements (made only in the final summer) suggested that variable management had achieved a quality improvement with increases in yields of digestible crude protein (19%) and digestible dry matter (9%) of the total forage produced in early summer. The improvements in the yields of digestible crude protein and digestible dry matter of the tall fescue component in late summer were even greater (28 and 19%, respectively). While advantages at other times of the year were expected to be smaller, the data suggested that the small loss in total yield was likely to be offset by increases in digestibility of available forage for grazing stock, especially in the critical summer period.

The performance of irrigated mixtures of tall fescue, ryegrass and white clover in subtropical Australia. 1. The effects of sowing mixture combinations, nitrogen and oversowing on establishment, productivity, botanical composition and persistence.

In the subtropics of Australia, the ryegrass component of irrigated perennial ryegrass (Lolium perenne) - white clover (Trifolium repens) pastures declines by approximately 40% in the summer following establishment, being replaced by summer-active C4 grasses. Tall fescue (Festuca arundinacea) is more persistent than perennial ryegrass and might resist this invasion, although tall fescue does not compete vigorously as a seedling. This series of experiments investigated the influence of ryegrass and tall fescue genotype, sowing time and sowing mixture as a means of improving tall fescue establishment and the productivity and persistence of tall fescue, ryegrass and white clover-based mixtures in a subtropical environment. Tall fescue frequency at the end of the establishment year decreased as the number of companion species sown in the mixture increased. Neither sowing mixture combinations nor sowing rates influenced overall pasture yield (of around 14 t/ha) in the establishment year but had a significant effect on botanical composition and component yields. Perennial ryegrass was less competitive than short-rotation ryegrass, increasing first-year yields of tall fescue by 40% in one experiment and by 10% in another but total yield was unaffected. The higher establishment-year yield (3.5 t/ha) allowed Dovey tall fescue to compete more successfully with the remaining pasture components than Vulcan (1.4 t/ha). Sowing 2 ryegrass cultivars in the mixture reduced tall fescue yields by 30% compared with a single ryegrass (1.6 t/ha), although tall fescue alone achieved higher yields (7.1 t/ha). Component sowing rate had little influence on composition or yield. Oversowing the ryegrass component into a 6-week-old sward of tall fescue and white clover improved tall fescue, white clover and overall yields in the establishment year by 83, 17 and 11%, respectively, but reduced ryegrass yields by 40%. The inclusion of red (T. pratense) and Persian (T. resupinatum) clovers and chicory (Cichorium intybus) increased first-year yields by 25% but suppressed perennial grass and clover components. Yields were generally maintained at around 12 t/ha/yr in the second and third years, with tall fescue becoming dominant in all 3 experiments. The lower tall fescue seeding rate used in the first experiment resulted in tall fescue dominance in the second year following establishment, whereas in Experiments 2 and 3 dominance occurred by the end of the first year. Invasion by the C4 grasses was relatively minor (<10%) even in the third year. As ryegrass plants died, tall fescue and, to a lesser extent, white clover increased as a proportion of the total sward. Treatment effects continued into the second, but rarely the third, year and mostly affected the yield of one of the components rather than total cumulative yield. Once tall fescue became dominant, it was difficult to re-introduce other pasture components, even following removal of foliage and moderate renovation. Severe renovation (reducing the tall fescue population by at least 30%) seems a possible option for redressing this situation.

The use of temperate species in the Australian subtropics.

Temperate species and tropical crop silage are the basis for forage production for the dairy industry in the Australian subtropics. Irrigation is the key resource needed for production, with little survival of temperate species under rain-grown conditions except for lucerne. Annual ryegrass (Lolium multiflorum), fertilised with either inorganic nitrogen or grown with clovers, is the main cool season forage for the dairy industry. It is sown into fully prepared seedbeds, oversown into tropical grasses, especially kikuyu (Pennisetum clandestinum) or sown after mulching. There has been a continual improvement in the performance of annual and hybrid ryegrass cultivars over the last 25 years. In small plot, cutting experiments, yields of annual ryegrass typically range from 15 to 21 t DM/ha, with equivalent on-farm yields of 7 to 14 t DM/ha of utilised material. Rust (Puccinia coronata) remains the major concern although resistance is more stable than in oats. There have also been major improvements in the performance of perennial ryegrass (L. perenne) cultivars although their persistence under grazing is insufficient to make them a reliable forage source for the subtropics. On the other hand, tall fescue (Festuca arundinacea) and prairie grass (Bromus willdenowii) cultivars perform well under cutting and grazing, although farmer resistance to the use of tall fescue is strong. White clover (Trifolium repens) is a reliable and persistent performer although disease usually reduces its performance in the third year after sowing. Persian (Shaftal) annual clover (T. resupinatum) gives good winter production but the performance of berseem clover (T. alexandrinum) is less reliable and the sub clovers (T. subterraneum) are generally not suited to clay soils of neutral to alkaline pH. Lucerne (Medicago sativa), either as a pure stand or in mixtures, is a high producing legume under both irrigation and natural rainfall. Understanding the importance of leaf and crown diseases, and the development of resistant cultivars, have been the reasons for its reliability. Insects on temperate species are not as serious a problem in the subtropics as in New Zealand (NZ). Fungal and viral diseases, on the other hand, cause many problems and forage performance would benefit from more research into resistance.

Manure and sorbent fertilisers increase on-going nutrient availability relative to conventional fertilisers

The key to better nutrient efficiency is to simultaneously improve uptake and decrease losses. This study sought to achieve this balance using sorbent additions and manure nutrients (spent poultry litter; SL) compared with results obtained using conventional sources (Conv; urea nitrogen, N; and phosphate–phosphorus; P). Two experiments were conducted. Firstly, a phosphorus pot trial involving two soils (sandy and clay) based on a factorial design (Digitaria eriantha/Pennisetum clandestinum). Subsequently, a factorial N and P field trial was conducted on the clay soil (D. eriantha/Lolium rigidum). In the pot trial, sorbent additions (26.2 g of hydrotalcite [HT] g P− 1) to the Conv treatment deferred P availability (both soils) as did SL in the sandy soil. In this soil, P delivery by the Conv treatments declined rapidly, and began to fall behind the HT and SL treatments. Addition of HT increased post-trial Colwell P. In the field trial low HT-rates (3.75 and 7.5 g of HT g P− 1) plus bentonite, allowed dry matter production and nutrient uptake to match that of Conv treatments, and increased residual mineral-N. The SL treatments performed similarly to (or better than) Conv treatments regarding nutrient uptake. With successive application, HT forms may provide better supply profiles than Conv treatments. Our findings, combined with previous studies, suggest it is possible to use manures and ion-exchangers to match conventional N and P source productivity with lower risk of nutrient losses.

Efficiency of rumen microbial protein synthesis in cattle grazing tropical pastures as estimated by a novel technique

The efficiency of microbial protein synthesis (EMPS) in cattle grazing a range of tropical pasture types was examined using a new method of intra-jugular infusion of CrEDTA to estimate urinary excretion of purine derivatives (PD). Seven pasture types were studied in south-east Queensland, Australia, over a 13-month period. These included native tropical grass (C4) pasture (major species Heteropogon contortus and Bothriochloa bladhii) studied in the early wet, the wet/dry transition and the dry season; introduced tropical grass (C4) pasture (Bothriochloa insculpta) in the mid wet season; two introduced tropical legume species (C3), (Lablab purpureus and Clitoria ternatea); and the temperate grass (C3) pasture, ryegrass (Lolium multiflorum). There was a large range in EMPS across pasture types: 26-209 g microbial crude protein (MCP)/kg digestible organic matter intake (DOMI). Estimated rumen degradable protein (RDP) supply (42-525 g/kg DOMI) was the major factor associated with EMPS across the range of pasture types studied. EMPS in steers grazing all tropical grass pastures was low (<130 g/kg DOMI) and limited by RDP supply. Negative linear relationships (P<0.05) between EMPS and both neutral detergent fibre (NDF) and acid detergent fibre (ADF) concentrations in extrusa were evident. However, non-fibre carbohydrate in extrusa, total non-structural carbohydrate concentration in plucked pasture leaf, rumen fluid and particle dilution rate, protozoal concentration in rumen fluid and rumen fluid pH were not correlated with EMPS. It was concluded that EMPS was well below 130 g MCP/kg DOMI when cattle grazed unfertilised, tropical grass pastures in south-east Queensland and that RDP was the primary limiting nutrient. High EMPS was associated with very high RDP, vastly in excess of RDP requirements by microbes