Subterranean Clover Cultivars Research Articles

Improving N2 fixation from the plant down: Compatibility of Trifolium subterraneum L. cultivars with soil rhizobia can influence symbiotic performance

Plant genotypes of Trifolium subterraneum L. (subterranean clover) were evaluated for differences in symbiotic N2 fixation with soil rhizobia, with the long-term aim of using plant selection to overcome sub-optimal N2 fixation associated with poorly effective soil rhizobia. Symbiotic performance (SP) was assessed for 49 genotypes of subterranean clover with each of four pure Rhizobium strains isolated from soil. Plants were grown in N free media in the greenhouse and their shoot dry weights measured and expressed as a percentage of dry weight with R. leguminosarm bv. trifolii WSM1325, the recommended commercial inoculant. Average SP with two Rhizobium strains (H and J) ranged from completely ineffective to 80% of potential for the subterranean clover genotypes. Two clover cultivars with high (cv. Campeda) and low (cv. Clare) SP values were investigated in more detail. Campeda typically fixed more N2 than Clare when inoculated with 30 soil extracts (4.2 vs 2.4 mg N2 fixed/shoot) and with 14 pure strains isolated from those soils (4.2 vs 2.2 mg N2 fixed/shoot). The poor performance of Clare could be attributed to interruptions at multiple stages of the symbiotic association, from nodule initiation (less nodules), nodule development (small, white nodules), through to reduced nodule function (N2 fixed/mg nodule) depending on the inoculation treatment. Through the careful use of subterranean clover genotypes by plant breeders it should be possible to make significant gains in the SP of future subterranean clover cultivars.

Effect of timing of forage conservation on forage yield and quality, seed yield and seedling regeneration of four subterranean clover (Trifolium subterraneum) cultivars

Swards of four cultivars of subterranean clover (Trifolium subterraneum L.) were cut at three different times to determine the effect on forage yield and quality, seed set and seedling regeneration in two successive seasons in southern New South Wales. The four cultivars of subterranean clover (Seaton Park LF, Junee, Goulburn and Clare) were cut on 23–25 September (early cut), 8–10 October (mid cut) or 22–23 October (late cut), to simulate an early silage, late silage or hay cut. Additional treatments imposed included either grazing or leaving the regrowth after cutting and raising the cutting height from 3 to 6 cm. Forage yields ranged from 3.5 to 9.3 t dry matter (DM)/ha in the first year and from 2.0 to 5.9 t DM/ha in the second year. Herbage yield was influenced by both cultivar and harvest time with the highest yields achieved with the mid cut. Lower forage yields at the later cut were attributed to losses due to respiration and decay under dense leaf canopies. Changes in forage quality were consistent across both years, with in vivo DM digestibility declining from 76–79% to 69–70% as cutting time was delayed. Crude protein fell from 22–24% to 14–17% over the same period, depending on cultivar. Seed yields in both years were influenced by both cutting time and cultivar with a positive relationship (R2 = 0.45–0.61) between herbage present in late spring after a period of regrowth and subsequent seed yield. The early flowering cultivar Seaton Park LF had the highest seed yield in both years and the more erect cultivar Clare had the lowest. Seed yields declined with later cutting time but increased by an average of 39% when the cutting height was raised from 3 to 6 cm. Seedling regeneration reflected seed yield responses with the largest seedling regeneration occurring in treatments cut early. The study found that forage conservation in early October is likely to yield more and be of higher quality than swards cut later in the season. Seed set is greatly reduced by all cutting strategies to levels unprofitable for seed harvesting but may be adequate for pasture regeneration.

Lucerne, phalaris, and wallaby grass in short-term pasture phases in two eastern Australian wheatbelt environments. 2. Effect of perennial density and species on subterranean clover populations and the relative success of 3 clover cultivars of different maturity

The effect of the density of 3 perennial species, phalaris (Phalaris aquatica L.), wallaby grass (Austrodanthonia richardsonii Kunth), and lucerne (Medicago sativa L.), on seed set, regeneration, and the relative competitiveness of 3 cultivars of subterranean clover (Trifolium subterraneum L.) was examined in 2 environments in the south-eastern Australian wheatbelt. Seed yields of subterranean clover were inversely related to perennial density at both sites over the first 2 years, the relationship varying with perennial species. Phalaris depressed the seed yield of clover more than lucerne and wallaby grass in the second and third year at equivalent densities. Clover seed yield was positively related to clover herbage yield in late spring at both sites, and inversely related to perennial herbage yield. Clover seed yield displayed an increasing linear relationship with the proportion of light reaching the clover understorey in spring, which in turn was inversely related to perennial density and perennial herbage yield. Clover seedling regeneration in mixed swards in autumn was positively related to the size of the summer seed bank, but negatively related to perennial density. Clover seedling survival following a premature germination at Kamarah was inversely correlated to the density of phalaris and lucerne in the sward. The relative competitiveness of the 3 subterranean clover cultivars varied between sites, with climatic conditions (rainfall and growing-season length) having a greater effect on the relative cultivar performance than companion perennial species or density. The later maturing subterranean clover cv. Goulburn became the dominant cultivar at the wetter site, constituting 72% of the seed bank, but declined to only 3–8% of the seed bank at the drier site. The proportion of the early flowering cultivar Dalkeith in the seed bank increased over time at the drier site and was highest (53%) in plots with the highest perennial density. We concluded that although perennial pasture species will depress clover seed yield and subsequent regeneration, these effects could be minimised by reducing perennial densities and exploiting variations in competitiveness between perennial species as identified in this study. Sowing earlier maturing subterranean clover cultivars would only be an advantage in increasing clover content in low-rainfall environments. The findings suggest that clover seed reserves and regeneration could also be increased by using grazing management to reduce the level of shading of clover by perennials, a factor associated with reduced clover seed yield.

The effects of subterranean clover phytoestrogens on sheep growth, reproduction and carcass characteristics

The aim of the present study was to evaluate the effect of a prolonged administration of phytoestrogens on sheep growth rate, female reproductive response, lamb carcass conformation and meat quality. To investigate these effects, two groups of Comisana sheep (24 females and 20 males, initial average live weights of 25.0 and 29.9 kg, respectively) were fed on subterranean clover (SC, phytoestrogen content = 0.8 mg/g of DM) and Italian ryegrass (C, non-oestrogenic control diet) for about one year. Feedstuffs were offered ad libitum and supplemented with maize grain and sunflower meal to maintain an adequate and similar energy and protein intake. The results demonstrated that the prolonged administration of the selected subterranean clover cultivars, with low formononetin content (lower than 10% of total isoflavones on dry basis), did not affect ewe reproduction but induced a significant improvement in animal weigh gain and, in males, good carcass and meat characteristics.

Comparing the potassium requirements of five pasture species

Potassium (K) deficiency is now common in sandy soils of south-western Australia and the K requirements of most pasture species grown in rotation with crops in the region are not known. In a glasshouse experiment using a K deficient yellow sand, we compared the K requirements of 5 pasture species now commonly grown in the region: French serradella (Ornithopus sativus Brot.) cv. Cadiz, yellow serradella (Ornithopus compressus Brot.) cv. Santorini, balansa clover [Trifolium balansae, classified as T. michelianum Savi var. balansae (Boiss)] cv. Paradana, 2 subterranean clover (Trifolium subterraneum L.) cultivars, cv. Seaton Park (subspecies subterraneum) and cv. Trikkala (subspecies yanninicum), and annual ryegrass (Lolium rigidum Gaud) cv. Wimmera. After 56 days of growth, above-ground growth was harvested and analysed for total K. Mitscherlich curves were fitted to yield of dried shoots, K concentration and K content (K concentration multiplied by yield) of the shoots. These fitted curves were used to determine the amount of K required to produce 75% of the maximum yield of dried shoots, K required to attain a K concentration in dried shoots of 25 g/kg, and K required to achieve a K content in dried shoots of 250 mg K/pot. Annual ryegrass and yellow serradella more effectively used indigenous soil K and applied K to produce dried shoots than the other species and cultivars, with the other species and cultivars requiring about 55% more applied K to produce 75% of the maximum shoot yield. The K content in dried shoots estimated the total K taken up from the soil, and annual ryegrass took up least K from soil, but it required least applied K to produce 75% of the maximum shoot yield, indicating it used the K it took up very effectively to produce shoots. Yellow serradella took up most K and, after annual ryegrass, it needed least applied K to produce the target yield of dried shoots. After yellow serradella, subterranean clover cv. Trikkala took up most K from soil, but it required nearly the most applied K to produce the target shoot yield so it was inefficient at using K taken up to produce the shoots. For all the other species and cultivars, K requirement was similar as assessed by yield or K content data. The critical concentration of K required for 90% maximum yield of dried shoots was about (g K/kg) 24 for balansa clover, 23 for both subterranean clover cultivars, 21 for yellow and French serradella, and 15 for annual ryegrass.

Occurrence ofPhytophthora clandestinaraces across rainfall zones in south-west Western Australia

Cultivars of subterranean clover (Trifolium subterraneum), Meteora and Denmark, sown in high rainfall zones in Western Australia were unique in their sensitivity to races of Phytophthora clandestina. The majority and most diverse of P. clandestina races occurred in the 700–1000 mm rainfall zone. Race 173 occurred across all rainfall zones ≥300 mm and race 177 in all zones ≥400 mm. Cultivar Denmark was highly resistant to races 001, 101, 141, 151 and 143, and moderately resistant to race 121, whereas cv. Meteora was highly resistant to race 151, moderately to highly resistant to races 001 and 101 and moderately resistant to races 121, 141 and 143. These differences are important in relation to the selection and breeding of cultivars for specific regions of Western Australia where particular races occur.

Characterization of Phytophthora clandestina races on Trifolium subterraneum in Western Australia

Phytophthora clandestina is a causal agent of root rot disease of subterranean clover in Western Australia (W.A). As a significant number of isolates of P. clandestina from W.A. could not previously be designated using existing differentials, a comprehensive set of subterranean clover (Trifolium subterraneum) cultivars was used as differentials to delineate a broader range of races of the pathogen. One hundred and one isolates of the pathogen collected from W.A. were screened on nine subterranean clover cultivars, of which seven were found to be useful as host differentials. A total of 10 races (in contrast to the five recognized previously) were defined and differentiated using octal nomenclature, presenting a clearer picture of the racial distribution of P. clandestina among W.A. isolates. Differences were found in the race populations between Australian states and are therefore important to the selection/breeding of cultivars for specific regions of Australia to counter the predominant race populations and for enforcing quarantine measures in relation to seed movements within and outside Australia. The octal nomenclature used provides a sound basis for follow-up studies and future race designations. Races 173 and 177 in this study were widely distributed and were the most common races in W.A., and together constitute 80% of the isolates characterized. While six of the seven host differentials were resistant to isolates belonging to race 001 and all were resistant to 000, it is of concern that only one differential was resistant to 157 and 173 and that none of the host differentials were resistant to 177. Our approach to P. clandestina race delineation is clearly conservative and is different from previous studies. The octal nomenclature we applied in this study is not only scientifically sound but also will facilitate rapid recognition and characterization of the races.

Genetic basis for and inheritance of resistance to Race 1 and Race 2 of Kabatiella caulivora in Trifolium subterraneum ssp. subterraneum and ssp. yanninicum

A study of a partial diallel involving crosses of seven cultivars of subterranean clover (Trifoliumsubterraneum ssp. subterraneum and ssp. yanninicum) was undertaken to determine the genetic basis of resistance to both Race 1 and Race 2 of Kabatiella caulivora at seedling and adult plant stages. Relationships between variance (Vr) and covariance (Wr) revealed that adult plant resistance to Race 1 and both seedling and adult plant resistance to Race 2 were controlled by simple additive and dominant gene effects, whilst seedling resistance to Race 1 was controlled by epistatic gene effects. Three of the four most resistant parents (cultivars Daliak, Denmark and Meteora) showed a dominant gene effect for adult plant resistance to Race 1. However, the other resistant parent, cv. Goulburn, and the most susceptible parent, cv. Woogenellup, both were controlled by recessive gene effects (i.e, gene effects conferred by recessive genes). Cultivar Denmark again showed dominant gene effects in relation to resistance to Race 2 at both seedling and adult plant stages, whilst recessive gene effects were evident in susceptible adult plants of cv. Daliak in response to Race 2. Estimates of genetic variance components confirmed that dominance variance (H1 and H2) was much greater than additive variance (D) and there were more dominant alleles (i.e., more alleles that had dominant effects) than recessive alleles (i.e., more alleles that had recessive effects) present across all the cultivars. An examination of segregation ratios in three F2 populations revealed that inheritance of adult plant resistance to Race 1 was controlled by a dominant gene in cv. Daliak and cv. Meteora, and by two dominant genes in cv. Denmark. The additional resistance gene present in cv. Denmark may well explain its apparent stable resistance to both races in the field. This study provides an insight into the genetic basis for and inheritance of resistance to this disease that will assist breeding programs aimed at improving resistance.

Leaf development and dry matter production of subterranean clover cultivars in relation to autumn sward management

Seeds of five cultivars of subterranean clover, together with one of white clover, were sown in a wide range of temperature regimes under both controlled environment and field conditions. Results were consistent across temperature regimes and showed that the first trifoliate leaf emerged after 230 °Cd for all subterranean clover cultivars and 309 °Cd for the white clover cultivar. For subterranean clover, exponential leaf appearance commenced after 434 °Cd at the six total leaf stage. At this time, subterranean clover can be defoliated without causing permanent physical damage to seedlings. The field study at Lincoln University showed that subterranean clover that germinated in March produced 44 kg DM/ha/day for 158 days to yield approximately 7 000 kg DM/ha by mid-September. Subterranean clover that germinated in May produced 15 kg DM/ha/day for 120 days to yield only 1 800 kg DM/ha by mid September. These results are discussed in relation to the time of autumn grazing management for subterranean clover, including extrapolation to other climatic areas of New Zealand. Key words: herbage yield, phyllochron, seedling establishment, thermal time, Trifolium subterraneum, T. repens, white clover

Subterranean clover in North Canterbury sheep pastures

The Hawarden basin is typified by dry, hot conditions over summer, which makes it difficult to retain ryegrass/white clover pastures on the lighter soils. These conditions have led to the introduction of better-adapted species such as the annual, subterranean clover. The mid-flowering cultivar 'Mt Barker' was first sown in the 1930s; then the earlyflowering 'Woogenellup' and late 'Tallarook' were used in the 1960s. The 'Mt Barker' type became dominant in these old pastures. The key factors for good subterranean clover growth were soil fertility (particularly P and S), space following dry summers for seedlings to re-establish, and moisture in the spring to maximise herbage yield. A visit to Australia in 1996 prompted the inclusion in new sowings of the late-flowering 'Leura' at high sowing rates (8-10 kg/ ha). It has taken 5 years for seed stocks to build to a level where we can identify good seedling strikes of 'Leura'. Two of the autumns over this period were very difficult and hindered normal establishment. Rainfall is erratic in North Canterbury, which affects the success of subterranean clover from one year to another. Dry summer conditions are optimal for subterranean clover strikes, as the pasture opens, and when the autumn break occurs, more than 2 000 subterranean clover seedlings/m2 can be produced. Autumn grazing management, when recovering from extreme drought, is critical because pasture must be spelled for 6 weeks to allow the subterranean clover to establish. However, if the autumn is wet, pasture cover must be controlled so that subterranean clover seedlings are not smothered. Set-stocking is best in spring to control pasture cover, but grazing should not be too hard during the flowering and seed set period. Subterranean clover seed yields of up to 250 kg/ha have been achieved from old pastures, but this is less seed than is produced under Australian conditions. Pasture renovation using the baker boot drill to re-introduce grasses and new cultivars of subterranean clover has been successful following very dry summers. Lamb growth rates have been excellent on subterranean clover-based pastures and these have increased the farm's meat production and reduced the cost of maintaining pastures. Key words: grazing management, North Canterbury, seedling establishment, seed set, subterranean clover

Abscisic acid added to the nutrient solution induces a water deficit‐like response in Trifolium subterraneum

SummaryThe response of subterranean clover (Trifolium subterraneum) to the addition of increasing concentrations of abscisic acid (ABA) in the nutrient solution (10−3, 10−2 and 10−1 mol m−3) was studied under growth room conditions. Six cultivars of contrasting yield capacity were compared.Plants were grown in Hoagland's solution until they had produced at least four fully developed leaves. ABA was then added and its effect on the fresh weight, leaf number and longest root length was determined, 1 day, 4 days, 7 days and 11 days after addition. The addition of ABA caused significant reductions in all the measured growth parameters, as well as a significant decrease in leaf water potential, which was dependent on the ABA concentration. The average growth reduction after 11 days under 10−1 mol m−3 ABA were within previously reported ranges for this crop under drought, in field conditions. The average leaf number, area of a fully developed leaf and the dry weight per plant of the six cultivars decreased by approximately 50%, whereas the root/shoot ratio increased by 80%.The variation and ranking for this treatment resembled closely those obtained for the same cultivars in field experiments. The cvs Clare, Nuba and Seaton Park, showed the best results under both control and ABA treated conditions. The correlation between the response to ABA in nutrient solution and previous water deficit studies raises the possibility of using this approach as an alternative way to quantify the drought sensitivity of subterranean clover cultivars.

Differences in symptom development in subterranean clover infected with Kabatiella caulivora Race 1 and Race 2 are related to host resistance

Clover scorch (Kabatiella caulivora) is a severe fungal disease of Trifolium spp. contributing to the collapse of pasture swards across southern Australia during warm, humid spring weather. Host plant responses associated with resistance to the disease were determined in 2 cultivars of subterranean clover (T. subterraneum) separately inoculated with K. caulivora Race 1 or Race 2. Germination of conidia of both races reached a maximum 5 days post-inoculation on cv. Woogenellup (susceptible to both races) and 4 days post-inoculation on cv. Daliak (resistant to Race 1 but susceptible to Race 2). Germ tube growth of Race 1 was inhibited on cv. Daliak and the percentage of conidia penetrating leaf surfaces was lowest on this race–cultivar combination. Susceptibility was characterised by large petiole lesions, with invasive hyphae extending through the mesophyll tissue into the pith and then through the phloem tissue of vascular bundles, eventually causing the petioles to collapse. Resistance was characterised by small, black lesions with invasive hyphae extending no further than the fourth layer of mesophyll cells. A suberin-based material was observed beneath infected mesophyll cells in the incompatible interaction, beyond which no further growth of hyphae occurred. Race 2 caused a faster rate of host tissue necrosis than Race 1 and also the breakdown of starch grains in uninvaded petiole tissues. Starch grains in plants infected with Race 1 were evenly distributed in uninvaded tissue. Sporulation was rarely observed in the incompatible interaction but was common in compatible interactions within 15 days post-inoculation. These responses to K. caulivora can now be used as a breeding tool in evaluating and selecting improved resistance to clover scorch disease among breeding lines of subterranean clover.

Effect of plant density on the winter production of annual clovers grown in monocultures

The small-seeded annual clovers, balansa and Persian, are often assumed to be poor winter producers. Their small seed size, of about 1 mg or less, and poor regeneration, possibly due to inappropriate grazing management in many instances, contributes to this perception. To test the hypothesis that early growth of these clovers is determined by the weight of germinating seed, as it is in subterranean clover, an experiment with 2 cultivars of subterranean clover, Leura and Trikkala, 2 cultivars of balansa clover, Paradana and Bolta, and 1 of Persian clover, Nitro Plus, was established in the field at Hamilton, western Victoria, at 6 sowing densities. The winter production at 2 additional sites, Lake Bolac and Streatham, in their third and second and third seasons, respectively, was also examined. Plant density varied from 30 to 37000 plants/m2 across sites and species. With equal weight of germinating seed per unit area at sowing, balansa and Persian clovers produced more herbage in winter than did the 2 subterranean clover cultivars Leura and Trikkala (P<0.05). Even though there was high correlation between seed weight and seedling weight across all species shortly after emergence (r2 = 0.99), by harvest time no differences in plant weight existed between any treatments growing at the same plant density. From this we conclude the following: (i) for the same weight of germinable seed per unit area, balansa and Persian clovers produced more dry weight per hectare than subterranean clover, because they had higher plant densities; (ii) there were no differences in dry matter production per hectare between species growing at similar plant densities by harvest time at the end of winter; (iii) it appeared that in winter the small-seeded species exhibited a higher relative growth rate than the 2 subterranean clovers.

Fate of annual pasture legumes seeds on a two-way thermo-gradient plate

Effects of diurnally alternating temperatures (5/5–45/45�C) were examined on a two-way thermogradient plate for non-dormant seeds of 14 annual pasture legumes. Seed fates (germination, temperature induced non-viability and temperature induced dormancy) were determined from daily seed counts over a 14-day period on-plate and a further 14 days after removal from the plate. These data clearly demonstrate the existence and extent of seed fates other than germination. Maximum dormancy occurred over a broad range of temperatures for seeds of Trifolium subterraneum L. var. subterraneum (Katzn. et Morley) Zohary and Heller cv. Woogenellup (40/35–30/25�C), Astragalus hamosus L. cv. Ioman (40/25–30/5�C) and Trifolium hirtum All. cv. Hykon (35/30–30/5�C). In contrast, maximum dormancy occurred over a narrow temperature range for Medicago truncatula Gaertn. var. truncatula cv. Paraggio (35/30–30/25�C), M. aculeata Willd. var. inermis (Aschers.) Heyn (30/25 and 30/20�C), and M. minima L. (35/20–35/5�C) seeds. Generally, non-viability was highest in all legumes when temperatures were greater than 35/30�C and not significantly different from the maximum value (100%) at temperatures greater than 40/30�C. At temperatures less than 35/30�C non-viability was less than the maximum (P<0.05) for seeds of all legumes, except M. truncatula cvs. Jemalong and Sephi, M. aculeata, and Vicia villosa ssp. dasycarpa (Ten.) Cav. cv. Namoi. Compared with the other subterranean clover cultivars germination levels T. subterraneum var. brachycalycinum (Katzn. et Morley) Zohary and Heller cv. Clare seeds were not significantly different from the maximum value at higher temperatures. In all annual medics, except M. aculeata germination was not significantly different to the maximum at temperatures greater than 25/20�C, with high germination (P<0.05) occurring at 30/25�C in Jemalong and M. scutellata (L.) Mill cv. Sava and at 30/20�C in Paraggio, Sephi, M. minima and M. polymorpha L. Maximum germination in Ioman and Hykon seeds occurred over a broader temperature range (35/20–15/10�C and 30/20–15/5�C, respectively) than in Namoi (30/15�C and 25/20–25/5�C). Probable implications of these data for field emergence of non-dormant seeds and soil seed reserves are discussed.

Productivity of pasture legumes and chicory in central New South Wales

Seven experiments were established across a range of environments (latitude 33°S) in central New South Wales to evaluate 52 legume cultivars and lines against currently recommended cultivars. Plots were grazed by either sheep or cattle after each harvest. Criteria for inclusion were that lines were either commercially available or in the process of being registered. Three experiments also included chicory. Sites had from 600 to 900 mm annual rainfall and were at altitudes of 440–1000 m. The 4-year program included the dry summer of 1990–91. White clover and subterranean clover were the most productive species over time. Among subterranean clovers, the subspecies subterraneum cultivars were more productive than the yanninicum or brachycalycinum subspecies. Other species such as balansa, Persian, strawberry, red and crimson clovers, lotus major and murex medic were more variable in production. These legumes often grew well in the establishment year, but failed to persist. Lucerne was in general, not as productive as white or subterranean clover. Caucasian clover and yellow serradella should be evaluated further as conclusive judgements could not be formed. Chicory was often the most productive species in the experiments, especially over the warmer 6 months of the year. It persisted under a 6-week harvest regime and during the drought year. The newer subterranean clover cultivars, Leura, Goulburn and Denmark all exceeded the production from the previously recommended cultivars, Woogenellup and Karridale, even though no major disease was evident in the later group. The lines 89820D and 89841E were sufficiently productive to warrant further evaluation and possible development as cultivars. In contrast, while Huia, Tahora, Bonadino and Tamar were often as productive as the recommended white clover cultivar Haifa, they were not consistently better. Where summer rainfall occurs and the annual rainfall exceeds 650 mm, the greater potential yield of white clover compared with subterranean clover justifies its use. However, no white clover cultivars survived the summer drought in 1990–91 as intact plants. Further work is needed to develop more drought-tolerant cultivars.

Variability of phenylalanine ammonia-lyase and peroxidase activities in leaves of subterranean clover is determined by their susceptibility to Kabatiella caulivora

Kabatiella caulivora is the causal organism of northern anthracnose or clover (Trifolium spp.) scorch disease. The activities of phenylalanine ammonia-lyase (PAL) and soluble peroxidase were determined in seedling leaves of two cultivars of subterranean clover (T. subterraneum) inoculated with race 1 or race 2 of K. caulivora. A small increase in activity of PAL was recorded in both cultivars 2–4 h post inoculation with either race. A second, large increase in PAL activity was observed only in the incompatible interaction (cv. Daliak inoculated with race 1), increasing 18-fold between 8 and 48 h post inoculation. Peroxidase activity in cv. Daliak increased rapidly within 2 h post inoculation with either race but was significantly higher in the incompatible interaction. Peroxidase activity in cv. Woogenellup increased by 4 h post inoculation with either race, but was significantly lower than that in cv. Daliak. Subsequent increases in peroxidase activity were recorded in both cultivars, however the levels remained constant in cv. Daliak infected with race 1, while activities in the other race-cultivar combinations decreased to control levels. It is hypothesised that the peak of activity of PAL at 48 h, and the rapid increase in peroxidase at 2 h are related to the race-specific resistance response of cv. Daliak to race 1 of K. caulivora, and that minor peaks of activity in the compatible interactions are general defence responses.

Proteome analysis of cultivar-specific interactions between Rhizobium leguminosarum biovar trifolii and subterranean clover cultivar Woogenellup.

Proteome analysis was used to identify proteins that are involved in the early stages of nodulation between the subterranean clover cultivar Woogenellup and the Rhizobium leguminosarum bv. trifolii strains ANU843 and ANU794. Strain ANU843 induces nitrogen-fixing nodules whereas strain ANU794 forms aberrant nodules on the roots of cv. Woogenellup that fail to develop beyond an early stage. Our aim was to identify proteins that might be involved in the early stages of nodulation over a 48 h period and to identify proteins that are differentially displayed during the interactions between the host and the two microbes. Proteome maps from control Woogenellup roots and inoculated roots were generated and compared at 24 and 48 h post inoculation. Over 1500 spots were resolved on all gels. Of the 16 protein spots that were differentally displayed or developmentally regulated, 10 were assigned putative identities. These included an alpha-fucosidase, several ethylene-induced proteins, a Cu/Zn superoxide dismutase, a hypothetical 16.5 kDa protein, tubulin alpha-chain, chaperonin 21 precursor and triosephosphate isomerase. Of the 22 constitutively expressed proteins spots examined, eight spots were assigned putative protein homologies through N-terminal sequencing and included several pathogenesis and stress-related proteins. The result may suggest that ethylene levels are upregulated during the early stages of infection but that this does not result in the induction of common pathogenesis-related proteins. The specific induction of alpha-fucosidase by ANU794 may be important in the nodulation failure phenotype of strain ANU794.

Effect of companion perennial grasses and lucerne on seed yield and regeneration of subterranean clover in two wheatbelt environments

Seed production of subterranean clover (Trifolium subterraneum L.) in mixtures with lovegrass (Eragrostis curvula (Schrader) Nees cv. Consol), cocksfoot (Dactylis glomerata L. cv. Currie), phalaris (Phalaris aquatica L. cv. Sirolan), danthonia (Austrodanthonia richardsonii (Cashm.) H.P. Linder, cv. Taranna), and lucerne (Medicago sativa L. cv. Aquarius) was compared with pure and degraded (invaded by annual volunteers) annual subterranean clover pasture at 2 sites (Junee and Kamarah) in the southern wheatbelt of New South Wales. Seed yields, clover seedlings in winter, and the change in the proportion of 3 subterranean clover cultivars (Dalkeith, Seaton Park, Goulburn) when grown with and without perennials were assessed. The effect of thinning the perennials to 10 plants/m2 on clover seed set was examined at the drier site. Seed production of subterranean clover in the mixtures was depressed by up to 50% compared with the pure and degraded annual swards. Initial clover seed poduction in the mixtures was at least 60 kg/ha even in the drought year at the wetter site (Junee), and >85 kg/ha at Kamarah, the drier site (seedling establishment at Kamarah failed in the drought year). Clover seed reserves in the following 2 years progressively increased to >300 kg/ha in the perennial swards at Junee but were <100 kg/ha by the end of the third year at Kamarah. In comparison, seed reserves in the pure clover and degraded annual swards were >650 kg/ha at Junee and >350 kg/ha at Kamarah. Reducing perennial density to 10 plants/m2 at the drier site increased clover seed yield about 3-fold in the first year compared with unthinned perennial swards. The increased seed yield was due to increased numbers of burrs set and increased seeds per burr and, in all perennial pasture treatments except lucerne, increased seed size. Clover seedling regeneration in 3rd and 4th year after sowing was substantially lower in the perennial-based mixtures than annual plots, with a significant (P < 0.05) positive correlation at both sites between clover seedling regeneration and seed bank size (1996, r2 = 0.46–0.64; 1997, r2 = 0.64–0.85). Following false breaks in early autumn, clover seedling populations were substantially higher in the pure and degraded clover treatments than in most perennial treatments. The proportion of the 3 cultivars present in the seed bank at the end of the pasture phase differed between sites but the sward type only influenced the proportion at the drier site. At the medium rainfall site, the later maturing cultivar Goulburn constituted 27–54% of the seed bank and the early flowering Dalkeith 25–46%, with unsown cultivars being insignificant ( <1%). At the low rainfall site, Dalkeith was the major component (33–52%) of the seed bank but the background population of unsown cultivars constituted 11–48%, the lowest proportion being in swards without a perennial component. The proportion of Goulburn was highest (23%) in the pure sward and lowest (10%) in lucerne and phalaris. It was concluded that subterranean clover could form relatively stable mixtures with perennials in medium rainfall environments, with clover populations increasing with time. In lower rainfall environments, clover seedling populations in perennial swards may be low due to reduced seed set and decreased seedling survival following early autumn rains. In these environments earlier maturing, hard-seeded cultivars are more likely to persist in mixtures and there is more potential for unsown cultivars to constitute a greater proportion of the sward. Decreasing perennial density offers scope for improving clover seed set and survival in these environments.

Diversity of Phytophthora clandestina isolated from subterranean clover in southern Australia : analysis of virulence and RAPD profiles

The virulence spectrum of 112 isolates of Phytophthora clandestina collected from 56 sites in four subterranean clover-growing states in southern Australia was determined using differential cultivars of subterranean clover. Five races were detected, with race 0 in all states except New South Wales, race 1 in all states, race 2 only in Victoria, race 3 only in New South Wales, and race 4 in Victoria and Western Australia. The level of genotypic diversity among the different P. clandestina populations was investigated using five RAPD primers. Among 30 bands amplified, only two were polymorphic. This enabled identification of four multilocus RAPD genotypes. Three of the four genotypes occurred in all four states. Races 2 and 3 occurred with RAPD genotypes 1 and 2 only whereas races 0 and 1 occurred in all four multilocus RAPD genotypes. These results indicate that the pathogenicity spectrum of P. clandestina can change rapidly.

Effect of phalaris (Phalaris aquatica L.) and lucerne (Medicago sativa L.) density on seed yield and regeneration of subterranean clover (Trifolium subterraneum L.)

A mixture of 3 subterranean clover (Trifolium subterraneum L.) cultivars (cvv. Goulburn, Seaton Park, and Dalkeith) was grown with 5–40 plants/m2 of phalaris (Phalaris aquatica L.) or lucerne (Medicago sativa L.) for 3 years at Wagga Wagga, NSW (147°21´E, 35°03´S). Clover seed yield was assessed each year, the number of regenerating clover seedlings was counted, herbage yield of clover and the perennials was measured, and the change in perennial density recorded. The influence of perennials on water availability in spring and light during clover seed set was also examined. The impact of perennial density on clover was assessed using regression analyses. In each of the 3 years, subterranean clover seed yield was negatively related to perennial density, but phalaris suppressed clover seed yield more than equivalent densities of lucerne in 2 of the years. Clover seed yield was positively related (R2 = 0.30–0.85) to clover biomass in spring in all 3 years (except for subterranean clover in phalaris in 1994) and to the proportion of total photosynthetically active radiation reaching the clover canopy beneath the perennial (R2 = 0.33–0.83) in 2 of the 3 years. There was a positive relationship between clover seedling regeneration in autumn and size of the summer seed bank in both years in lucerne (R2 = 0.40–0.76) and in 1 year in phalaris (R2 = 0.76) and a negative association between perennial density and clover seedling regeneration. The rate at which the surface profile (0–37 cm) dried in spring was independent of phalaris and lucerne density over the 3 years and did not differ from that of pure clover. The late season clover cultivar, Goulburn, constituted between 57% and 79% of the seed bank following seed set, substantially more than the earlier flowering cultivars, Seaton Park and Dalkeith. Neither perennial density nor species changed the relative competitiveness of the 3 cultivars. The high seed yield of Goulburn and the lack of a perennial-induced change in surface soil water in spring suggest that growing perennials in association with subterranean clover does not increase the level of moisture stress during clover seed set. The good performance of Goulburn is attributed to superior competitive ability for light in late spring. The basal area occupied by lucerne and phalaris increased with perennial density in each of the first 2 years. However, in the third year, phalaris basal area was similar at all densities. The findings suggest that sowing low densities of lucerne should assist in promoting seed yield and regeneration of subterranean clover. Management of phalaris, which has the capacity to substantially increase plant size at low densities, may require additional strategies such as more frequent grazing in spring.