Yield,root morphology and chemical composition of two pasture legumes as affected by lime and phosphorus applications to an acid soil

Summary Effects of increasing rates of lime (0, 900, 1725, and 3000 kg Ca(OH)₂/ha producing soil pH of 4.0, 4.7, 5.1 and 5.6) and P (50, 150, 250 and 350 kg P/ha) on top and root yield, root morphology and chemical composition of lotus (Lotus pedunculatus Cav.) and white clover (Trifolium repens L.), were studied, using an acid soil in a greenhouse experiment. Increasing rates of applied lime and phosphate resulted in substantial increases in top yields of both species but concomitant increases in root yield were small. In the unlimed soil, lotus out-yielded (tops and roots) white clover at all P levels. However, in the three limed treatments, white clover clearly out-yielded lotus. Yield response curves to applied P levelled off at the two highest lime rates for lotus but not for white clover. Nodulation and N content of white clover increased significantly with increasing lime applications, but for lotus there was a significant decrease in nodulation at the highest lime rate. Increased P rates had a small stimulatory effect on nodulation in both species. Of the total root weight, the percentage contribution of the tap and primary lateral root fractions was smaller and that of the secondary plus tertiary lateral roots was greater for lotus than for white clover although root length per unit weight tended to be larger for white clover at the two highest lime rates. Furthermore, lotus possessed longer and more numerous root hairs than white clover. Lime applications significantly decreased the percentage contribution of the tap and primary lateral roots to the total root weight and increased the percentage contribution of the secondary plus tertiary lateral roots. Al and Mn contents of tops and roots of both species decreased with increasing lime rates. There was a highly significant negative correlation between relative yield and Al content of lotus and white clover tops. In comparison with the limed treatments, in the unlimed treatments a greater percentage of total P, Al, Mn and N content accumulated in the roots of both species. In addition, lotus accumulated a much greater percentage Al in its roots than white clover.     

The potential of legumes as a nitrogen source for the reclamation of derelicht land

Summary White clover (Trifolium repens) growing in association with grasses on colliery spoil amended with lime and complete fertilizer proved to be an effective nitrogen source for the developing ecosystem. Nitrogen transfer from the clover to an associated grass became aparent within 22 months of sowing.     

White clover cryptic virus‐1 in New Zealand and eastern Australia

The distribution and abundance of cryptic viruses (Partitiviridae) in natural and agricultural systems have received little attention. White clover cryptic virus‐1 (WCCV‐1) was detected infecting white clover plants and seed growing in New Zealand, but was not detected in field collections and in only two of 114 seedlings grown from seed collected in eastern Australia using enzyme‐linked immunosorbent assay and RT‐PCR. WCCV‐1 was detected at high incidences (14–48%) in the cultivars Huia, Kopu II and Tahora and at low incidences (0–7%) in Aran, Haifa, Ladino, Siral and Sustain white clovers. WCCV‐1 remained viable in seed stored for 50 years. The results are compared with another group of obligate seed‐transmitted pasture microbes: the fungal endophytes of perennial grasses.     

Subterranean clover decline in permanent pastures in north-eastern Victoria

Experimental sites were established at two locations in north-eastern Victoria to define factors limiting the establishment and growth of Trifolium subterranean L. (subterranean clover). Liming the soil, seed inoculation and fungicide application were used in renovating subterranean clover pasture on two acidic soils (Longwood: brown/grey sandy loam DY 3.14 and Seymour: grey brown light clay DY 3.22, Northcote classification) with mean annual rainfall of 650 mm and 600 mm respectively. Soil acidity, low available soil phosphorus and plant disease were identified as factors limiting clover yield on these soils. Significant yield responses to lime (35–140%) were obtained with subterranean clover at both sites, with corresponding decreases in Al in the 0–10 cm soil horizon. Liming the soil, when combined with seed inoculation, increased the number and effectiveness of root nodules at both sites. Soil P available for plant growth was low at both sites (6.1 and 8.4 μg g⁻¹) resulting in sub-optimal P concentrations in the clover herbage (45 mmol kg⁻¹ at Longwood). Levels of root disease were low but Aphanomyces euteiches and Phytophthora clandestina (causal agents of lateral and tap root rot) were detected frequently on roots. Application of fungicide resulted in higher dry matter yields (p=0.05) at both sites. An assessment of the relative contributions of these limiting factors and the benefits to be obtained from better management would provide a clearer picture of the profitability and sustainability of this farming system.     

Influence of pollinator abundance and flower visitation on seed yield in red clover

Red clover (Trifolium pratense) is an important forage crop in temperate regions. However, the seed yield in red clover, especially in tetraploid cultivars, constrains the commercial success of this crop. The relationship between seed yield and several flower and inflorescence characteristics has been investigated previously, but up to now no clear link has been identified. The central questions in this article are as follows: (1) Whether nectar production and composition are correlated with seed yield? (2) Which is the main pollinator found on red clover in Flanders? (3) Whether pollinator preferences can explain differences between high and low seed-yielding cultivars and genotypes? We found that Bombus pascuorum was the most abundant pollinator on red clover in the region considered. Nectar volume, nectar composition or pollinator preference did not explain the variation in seed yield between genotypes, nor between diploid and tetraploid cultivars. To explain seed yield differences among red clover genotypes and ploidy levels better, we believe that plant-related characteristics like problems with meiosis, low pollen or seed viability or embryo abortion need to be investigated.     

Seed-borne cucumber mosaic virus infection of subterranean clover in Western Australia

In Western Australia, infection with cucumber mosaic virus (CMV) was widespread in all three subspecies of subterranean clover (Trifolium subterraneum) growing in plots belonging to the Australian National Subterranean Clover Improvement Programme. Seed-borne CMV was detected in seed harvested in 1984–1986 of 18/25 cultivars from two collections of registered cultivars; seed transmission rates ranged up to 8.8%. Seed samples from CMV-inoculated plants of 11 cultivars transmitted the virus to 0.5–8.7% of seedlings. Seed transmission rates greater than 5% were obtained only with cvs Enfield, Green Range and Nangeela. CMV was not detected in seed harvested in 1975–1981 from one of the registered cultivar collections, in 17 commercial seed stocks from 1986 or in a survey of subterranean clover pastures.
Symptoms in subterranean clover naturally infected with CMV included mottle, leaflet downcurling and dwarfing but severity varied with cultivar and selection. CMV isolates from different sources varied in virulence when inoculated to subterranean clover; two (both from subterranean clover) were severe, two moderate and three (including one from subterranean clover) mild. In pot tests, CMV decreased herbage production and root growth (dry wts) of cv. Green Range by 49% and 59% respectively. In spaced-plants growing in plots, CMV decreased herbage production and root growth of cvs Green Range and Northam by 59–630 and seed production of cv. Green Range by 45%. In rows sown with infected seed, aphid spread increased infection levels to 75% in cv. Green Range and 44% in cv. Esperance and losses in herbage production of 42% and 29% respectively were recorded.
CMV isolated from subterranean clover included isolates from both serogroups.     

Studies on Taxonomic Relationships of Mycoplasma–like Organisms by Southern blot Analysis

Chromosomal DNA fragments from the mycoplasma-like organisms (MLOs) associated with American aster yellows, apple proliferation, clover phyllody, and vaccinium witches' broom were cloned. Several MLO-specific fragments from each of these four isolates and a sequence from the 16S rRNA gene of an aster yellows MLO were used in Southern blot hybridizations to investigate the taxonomic relationships of 26 pathologically and geographically diverse MLOs. These MLOs were divided into four categories according to the symptoms induced in periwinkle. Genotypically, these isolates represented four groups (16S RFLP groups) of a classification based on restriction fragment length polymorphisms (RFLP) and sequencing data of the 16S rRNA gene. Probes from three isolates of one symptom category hybridized with isolates from all symptom categories. This result indicates that classification of MLOs by symptomatology does often not coincide with genetic relationships. The hybridization results confirmed the findings, of the 16S RFLP classification that most MLOs from herbaceous plants, especially those inducing virescence in periwinkle, are interrelated. These isolates, which were assigned to one 16S RFLP group, could be further differentiated in this study. Itcould be shown that aster yellows, clover phyllody, stolbur, and safflower phyllody and sandal spike are caused by distinct MLOs. The MLOs associated with apple proliferation, vaccinium witches' broom, and witches' broom of lime as well as two isolatesfrom, stone fruits could also be recognized as distinct organisms.     

Effect of soil moisture on the response of subterranean clover to lime

Summary A pot experiment was carried out using a soil high in aluminium to investigate the relationship between the dry matter responses of subterranean clover to lime at two soil moisture levels. Subterranean clover, which is reputed to be tolerant to high aluminium levels in the soil, showed an increase in the dry weight of plant tops of 50% when lime was added on the low moisture treatments. By contrast the response on the high moisture treatment was only 5.6%.     

Effect of Nitrogen Supply on the Grass and Clover Components of Simulated Mixed Swards Grown under Favourable Environmental Conditions I. Carbon Assimilation and Utilization

Simulated mixed swards of Perennial Ryegrass (Lolium perenneL.) cv. S23 and White clover (Trifolium repens L.) cv. S100were grown from seed under a constant 20 °C day/15 °Cnight temperature regime and their growth and carbon economyexamined. The swards received a nutrient solution daily, whichcontained either High (220 mg l^–1) or Low (10 mg l^–1)nitrate N. Rates of canopy photosynthesis and respiration, and final drymatter yields were similar in the two treatments although theproportions of grass and clover differed greatly. The Low-Nswards were made up largely of clover. The grass plants in theseswards had high root: shoot ratios and low relative photosyntheticrates – both signs of N deficiency – and were clearlyunable to compete with the vigorously growing Low-N clover plants.These had higher relative growth rates and dry matter yieldsthan their High-N counterparts. In the High-N swards clovercontributed around 50 per cent to the sward dry weight throughoutthe measurement period despite having a smaller proportion ofits dry weight in photosynthetic tissue (laminae) than grassover much of it. The latter was compensated for, initially bya higher specific leaf area than grass, and later by a higherphotosynthetic rate per unit leaf weight. The results are discussedin relation to observed declines in the clover content of swardsafter the addition of nitrogen fertilizer in the field. Trifolium repens, white clover, Lolium perenne, perennial ryegrass, nitrogen, photosynthesis, carbon balance     

Effect of pollen competition and stigmatic receptivity on seed set in white clover (Trifolium repens L.)

The effect of competition between incompatible and compatible pollen grains on the seed production of white clover was studied. Stigmatic receptivity was also studied. A selection line of white clover that has red leaves was used as a pollen donor and as a genetic marker to allow determination of the pollen donor responsible for ovule fertilisation. Results show that incompatible pollen did not inhibit compatible pollen grains from fertilising ovules and producing seeds, although it did slightly impair seed yield. At temperatures of 20/10°C (day/night) stigmas of white clover remained receptive to additional pollination up to 32 h after an initial compatible pollination and 40 h after an incompatible pollination. The results imply that factors other than inadequate pollination and the self-incompatibility system are responsible for failure of white clover to attain its potential seed yield. Received: 31 August 1999 / Revision accepted: 28 February 2000     

SEEDLING RECRUITMENT LIMITATION IN WHITE CLOVER (TRIFOLIUM REPENS; LEGUMINOSAE)

White clover (Trifolium repens L.) is a stoloniferous clonal legume. Dynamics of white clover populations are controlled by the recruitment of both seedlings (genets) and node production (ramets). To understand the role of seedling dynamics in natural populations of white clover, the recruitment and mortality of seedlings in a lawn and pasture clover population were followed and the roles of biotic and abiotic factors in seedling recruitment and survival were assessed. Seedling recruitment was several orders of magnitude less than vegetative recruitment in both lawn and pasture populations. However, at least several new genets are added to these populations on a yearly basis. In both locations, physical disturbances may open sites for seed germination, as disturbed sites had higher seed germination rates than undisturbed sites. However, these sites were not favorable for long-term establishment due to such factors as frost upheaval. Therefore, there are trade-offs in safe site attributes for seed germination and establishment that strongly limit recruitment in natural populations of white clover.     

Flowering Sequence and the Ordering of Impermeability Breakdown in Seed of Subterranean Clover (Trifolium subterraneum L.)

The pattern of breakdown of seed coat impermeability of seedwithin the burrs of two cultivars of subterranean clover (Trifoliumsubterraneum L.) was closely associated with relative differencesin flowering sequence within the inflorescence. The patternof seed weight variation showed very little relationship withthe pattern of breakdown, while that for seed moisture contentshowed no relationship. Within a burr it was almost invariably the seed which developedfrom the latest flower within the inflorescence which was thefirst to become permeable. An ordering of the timing of breakdownwas evident only within the burr; there was no evidence fora pattern of breakdown of impermeability between the differentburrs along the runner. Trifolium subterraneum L., subterranean clover, seed coat impermeability, seed weight, seed moisture, hardseededness     

Effects of phosphate fertilization,lime amendments and inoculation with VA-mycorrhizal fungi on soybeans in an acid soil

Soybeans [Glycine max (L.) Merr. cv. Essex] were grown in nonsterile acid (pH. 5.2) infertile Wynnville silt loam (Glossic Fragiudult) in a glasshouse. The effects of P fertilization and lime were determined by inoculation with two VAM-fungi (VAMF): Glomus fasciculatum (Gf) and Glomus etunicatum (Ge). An important factor affected by the interaction between applied lime (soil acidity), applied P, and VAMF inoculation was the soil Al. Five application rates of P as KH₂PO₄ and three rates of lime were tested. Potassium was equalized with KCl (muriate of potash). P-efficiency (g seed/mg P kg^-1 soil) by vesicular-arbuscular mycorrhiza (VAM) was maximal at 20 mg P kg^-1 soil at all lime and VAMF treatments. VAMF inoculation increased plant survival and protected the soybeans from leaf scorch, thereby substituting for the effects of lime and P. The Ge inoculum was superior in ameliorating leaf scorch in the nonlimed soil. The Gf inoculum required more lime and P than the Ge inoculum to increase seed yield relative to the noninoculated controls containing only native VAMF. Both inocula increased root Al uptake and extractable soil Al in the acid soil without apparent adverse effects on root or shoot. The ability of the VAMF inocula to enhance the efficiency of applied P and decrease seed Cl concentration was increased by lime. Seed yield (Y) was negatively related to seed Cl concentration (X) where Y=aX^-b. Both VAMF inoculation and lime application reduced this negative relationship and may have increased the tolerance to both Cl and soil Al.     

Effect of Nitrogen Supply on the Grass and Clover Components of Simulated Mixed Swards Grown under Favourable Environmental Conditions II. Nitrogen Fixation and Nitrate Uptake

Simulated mixed swards of Perennial Ryegrass (Lolium perenneL.) cv. S23 and White clover (Trifolium repens L.) cv. S100were grown from seed under a constant 20 °C day/15 °Cnight temperature regime and harvested at intervals over and88 d growht period. The swards received a nutrient solutiondaily, which was either High (220 mg l^–1) or Low (10 mgl^–1) in nitrate N. The nitrate was labelled with the ¹⁵Nisotope. An acetylene reduction assay was carried out on eachsward just prior to harvest. Rates of acetylene reduction agreed qualitatively with the ^l5Nanalyses but absolute values did not match (assuming a 4:1 C₂H₄:N₂ratio) and errors in the acetylene assay are discussed. In theLow-N swards clover relied almost entirely on symbioticallyfixed N₂, fixing more than ten times as much as the High-N cloverplants. In the Low-N treatment the grass was N-deficient despiteobtaining much more nitrate per unit root dry weight than clover.In the High-N swards, however, clover took up more nitrate perunit root weight than grass. The High-N clover plants also fixedsome N₂ and maintained a higher total-N content than grass throughoutthe period. There was no evidence of transfer of symbioticallyfixed N from the clover to the grass in either treatment. Trifolium repens, Lolium perenne, nitrate, nitrogen fixation, ¹⁵N, acetylene reduction     

QTL analysis of seed yield components in red clover (Trifolium pratense L.)

Cultivars of red clover (Trifolium pratense L.), an important forage crop in temperate regions, are often characterised by an unsatisfactory level of seed yield, leading to high production costs. This complex trait is influenced by many components and negatively correlated with other important traits, such as forage yield or persistence. Therefore, seed yield has proven to be difficult to improve. Thus, the objectives of this study were to assess association among seed yield components and to provide the basis for identifying molecular markers linked to QTLs for seed yield components to assist breeding for improved red clover cultivars. A total of 42 SSR and 216 AFLP loci were used to construct a molecular linkage map with a total map length of 444.2 cM and an average distance between loci of 1.7 cM. A total of 38 QTLs were identified for eight seed yield components. The traits seed number per plant, seed yield per head, seed number per head, head number per plant and percent seed set were highly correlated with seed yield per plant, and QTLs for several of these traits were often detected in the same genome region. Head number per plant may present a particularly useful character for the improvement of seed yield since it can easily be determined before seed maturity. In addition, two genome regions containing four or five QTLs for different seed yield components, respectively, were identified representing candidate regions for further characterisation of QTLs. This study revealed several key components which may facilitate further improvement of seed yield. The QTLs identified represent an important first step towards marker-assisted breeding in red clover.     

Yield responses of two ryegrass and two white clover varieties grown in contrasting mixtures with applications of phosphate at different depths and on different soils

To validate earlier findings using ³²P in upland soil and at different depths in lowland soil, ryegrass varieties S.23 or Titania were grown in mixtures with white clover varieties S.184 or Olwen. Unlabelled phosphate (0 or 43 kg P/ha) was applied to the surface or at 30 cm depth in lowland soil, and dry matter yields of grass and clover were measured. In June, on lowland soil, the greatest yield of clover was obtained by surface application. The most productive mixture was S.23/Olwen, although in August Titania outyielded S.23. By October S.23 again outyielded Titania. These results validated the isotopically discovered interactions between phosphate depth and grass/clover mixtures. On upland peat and mineral soil, 0 or 43 kg P/ha was surface applied to the same varietal mixtures. In May, phosphate application to mineral soil slightly decreased clover presence, while clover was increased by mixture with Titania rather than S.23. In July, no significant phosphate effects were found, but in October phosphate increased clover yield and, at least on mineral soil, S.23/Olwen responded most.     

Preliminary studies on sap-transmissible viruses of red clover (Trifolium pratense L.) in England and Wales

Red clover plants, collected from nine widely separated permanent pastures in England and Wales, were tested for sap-transmissible viruses. Viruses were identified by the symptoms they caused in test plants, by electron microscopy, and by serological tests. Of the 265 plants tested 14% were infected. Only pea mosaic virus was common and widespread; it was found in 8% of the plants, and in seven of the fields. Other viruses isolated were arabis mosaic, bean yellow mosaic, red clover mottle, and red clover vein mosaic; only red clover mottle virus produced diagnostic symptoms in red clover. No viruses were detected in seedlings grown from seed from eighty-nine commercial seed crops. Attempts to transmit red clover mottle virus by the Collembolan Sminthurus viridis L., which is common on red clover, failed.     

Effect of lime,phosphorus and mycorrhizal fungi on growth,nodulation and nitrogen fixation by white clover (Trifolium repens) grown in UK hill soils

Summary The responses of white clover (cv NZ Grasslands Huia grown in four UK hill soil types) to additions of lime and P, to inoculation with Rhizobium and mycorrhizal fungi, and to differences in soil water status were assessed in pot and field experiments. With a deep peat soil in pots, shoot production, nodulation and N fixation by clover were increased by 160, 130 and 85% respectively following inoculation with mycorrhiza, but in the field, despite a doubling of root infection, there was no response in growth. On a brown earth soil in the field inoculation with one endophyte (Glomus mosseae L1) out of four tested depressed production of white clover shoots by 42% but enhanced that of leeks (Allium porrum) by 50%; the others were without effect. With dry peaty podzol and brown earth soils in pots, clover shoot production was highest with added P when a water holding capacity of 80% was maintained, but roots from the latter had only 2.6 compared to 68 nodules per plant from the former. Further work is required to explain poor nodulation in the brown earth soils.     

Effect of inoculum rate on mycorrhizal growth responses in pot-grown onion and clover

Summary White clover and onion plants were grown from seed in pots of sandy loam above pads of mycorrhizal inoculum soil at 0.17–1.40 g/pot (equivalent to 250–2000 kg/ha) and harvested on four occasions. In sterilized soil increasing inoculum rates increased the onset and size of the mycorrhizal growth response of white clover. In unsterilized soil the indigenous mycorrhizal fungi greatly stimulated growth of both clover and onion. Nevertheless, all mycorrhizal inoculum rates further stimulated shoot growth in onion (92% increase over all harvests), while only the highest inoculum rate significantly stimulated clover growth (52% increase).     

Selective inhibitors of germination of legume seeds in activated sludge compost

Water extracts of the compost produced from activated sludge and coffee residue were found to be selectively inhibitory to seed germination of some legumes. Germination rate of white clover (Trifolium repens L.), red clover (Trifolium pratense L.) and alfalfa (Medicago sativa L.) seeds were reduced to 2, 29 and 73% of the control, respectively, by water extracts of the compost (20 g l^–1). However, the extracts did not show any inhibition to seed germination of sorghum (Sorghum bicolor Moench), African millet (Eleusine coracana Gaertn.), and Komatsuna (Brassica rapa L.) at the same concentration. The inhibitors in the compost extracts were separated by ion-exchange chromatography and reverse-phase high performance liquid chromatography (HPLC) and the inhibitory activities of seed germination were tested with white clover seeds. Five inhibitors were isolated and identified as 3,4-dichlorophenylacetic acid (3,4-DCP), 3,4-dichlorobenzoic acid (3,4-DCB), 3,4,5-trichlorophenylacetic acid, 3,4,5-trichlorobenzoic acid and mono-2-ethylhexylphthalate by ¹H-, ¹³C-NMR spectroscopy and mass spectrometry. The inhibitory activities of some authentic chemicals of the inhibitors and the related compounds were compared. The results indicated that the main inhibitor in the compost could be 3,4-DCB, which was contained at the concentration of 6.58 mg kg^–1 compost and showed the strongest inhibitory effect on seed germination of white clover among the tested compounds.