Oxygen deficiency affects carbohydrate reserves in overwintering forage crops

Anaerobic conditions developing under an ice cover affect winter survival and spring regrowth of economically important perennial crops. The objective was to compare, during a prolonged period of low (<2%) O2 at low temperature, the concentration of carbohydrates of four plant species contrasting in their resistance to oxygen deficiency. Four perennial forage species, lucerne (Medicago sativa L.), red clover (Trifolium pratense L.), timothy (Phleum pratense L.), and cocksfoot (Dactylis glomerata L.) were subjected to a progressively developing oxygen deficiency stress by enclosing potted plants in gas-tight bags in late autumn for overwintering in an unheated greenhouse. Timothy was previously reported to be more resistant to oxygen deficiency than the three other species. Non-structural carbohydrates increased and remained at a higher concentration in timothy than in the other three species under low O2 concentration. Concentrations of sucrose, fructose, glucose, and fructans increased in response to oxygen deficiency in timothy, whereas the concentration of soluble sugars decreased under the same conditions in lucerne, red clover, and cocksfoot. The gene expression of glyceraldehyde-3-phosphate dehydrogenase increased in response to low oxygen concentration in oxygen deficiency-sensitive lucerne while it remained unchanged in the oxygen deficiency-resistant timothy. It is concluded that timothy maintains higher carbohydrate reserves under oxygen deficiency, a specific feature that could favour its winter survival and spring regrowth.     

Host range and overwintering sources of bean leaf roll and pea enation mosaic viruses in England

Bean leaf roll virus (BLRV) and pea enation mosaic virus (PEMV) were each transmitted by Acyrthosiphon pisum (Harris) to fifteen of thirty species of legumes in the glasshouse; eleven species were susceptible to both viruses. The only biennial or perennial species infected by BLRV were hop trefoil (Medicago lupulina L.), lucerne (M. sativa L.) and red clover (Trifolium pratense L.), but naturally infected sainfoin (Onobrychis viciifolia Scop.) and white clover (T. repens L.) were found. The only perennial species infected with PEMV in the glasshouse was kidney vetch (Anthyllis vulneraria L.). Eggs of A. pisum, which seems to be the main vector of BLRV and PEMV in England, were found in winter on several species of cultivated perennial legumes, most on lucerne, fewest on white clover. In spring, more viviparae of A. pisum were found on lucerne than on other perennial legumes, and many on lucerne, but few on red or white clover, were infective with BLRV. Lucerne is probably the main overwintering source of BLRV in areas where lucerne is common, but elsewhere red and white clovers are probably as important. No aphid collected from perennial legumes between 1965 and 1968 was infective with PEMV, but this virus can overwinter in common vetch (Vicia sativa L.). Lucerne infected with BLRV was usually symptomless or showed only transient mild yellowing of young leaves. Lucerne plants showing vein-yellowing, similar to that previously reported as a symptom of BLRV, were possibly infected with an aberrant strain of BLRV but more probably with BLRV and another aphid-transmitted agent. Inoculations from lucerne with vein-yellowing symptoms sometimes caused vein-yellowing, and sometimes typical BLRV-symptoms, in crimson clover (Trifolium incarnatum L.).     

Species variation in the fixation and transfer of nitrogen from legumes to associated grasses

Summary Three legume species (alfalfa, red clover, and birdsfoot trefoil) in combination with five grass species (timothy, bromegrass, red fescue, tall fescue, and orchardgrass) were used to study N transfer in mixtures, using the 15_N dilution technique. The advantage of grass-legume mixtures was apparent. Total herbage and protein yields of grasses in mixtures were higher than those alone, especially at the later cuts. This benefit of mixed cropping is mainly due to N transfer from legumes to associated grasses. N₂-fixation and N transfer by alfalfa rated highest, red clover intermediate, and birdsfoot trefoil lowest. The importance of each pathway of N transfer from legumes appeared to differ between species. Alfalfa and red clover excreted more N than trefoil, while the latter contributed more N from decomposition of dead nodule and root tissue. The greatest advantage from a grass-legume mixture, with respect to the utilization of N released from the legume, varied with early maturing tall fescue (Kentucky 31), orchardgrass (Juno), and bromegrass (Tempo), to intermediate timothy (Climax), and least with late maturing red fescue (Carlawn). Contribution no. 817 of the Ottawa Research Station.     

Genome mapping of white clover (<Emphasis Type="Italic">Trifolium repens</Emphasis> L.) and comparative analysis within the Trifolieae using cross-species SSR markers

Allotetraploid white clover (Trifolium repens L.), a cool-season perennial legume used extensively as forage for livestock, is an important target for marker-assisted breeding. A genetic linkage map of white clover was constructed using simple sequence repeat (SSR) markers based on sequences from several Trifolieae species, including white clover, red clover (T. pratense L.), Medicago truncatula (Gaertn.) and soybean (Glycine max L.). An F₁ population consisting of 179 individuals, from a cross between two highly heterozygous genotypes, GA43 and Southern Regional Virus Resistant, was used for genetic mapping. A total of 1,571 SSR markers were screened for amplification and polymorphism using DNA from two parents and 14 F₁s of the mapping population. The map consists of 415 loci amplified from 343 SSR primer pairs, including 83 from white clover, 181 from red clover, 77 from M. truncatula, and two from soybean. Linkage groups for all eight homoeologous chromosome pairs of allotetraploid white clover were detected. Map length was estimated at 1,877 cM with 87% genome coverage. Map density was approximately 5 cM per locus. Segregation distortion was detected in six segments of the genome (homoeologous groups A1, A2, B1, B2, C1, and D1). A comparison of map locations of markers originating from white clover, red clover, and alfalfa (M. sativa L.) revealed putative macro-colinearity between the three Trifolieae species. This map can be used to link quantitative trait loci with SSR markers, and accelerate the improvement of white clover by marker-assisted selection and breeding. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Infection Biology of Bacterial Wilt of Forage Grasses

Infection of perennial ryegrass (Lolium perenne L.), Italian ryegrass (Loliummultiflorum L.), and timothy (Phleum pratense L.) by Xanthomonas campestris pv. graminis and Xanthomonas campestris pv. phlei occurred mainly via wounds rather than natural openings. Nevertheless, bacteria were detected by isolation and immunofluorescence in plants sprayed with the pathogen without prior wounding and in plants in which intact ears had been dipped in inocula. High concentrations of bacteria were observed around the stomata of perennial ryegrass and timothy by scanning electron microscopy 48 h after inoculation. Perennial ryegrass and Italian ryegrass and individual plants of ryegrass and timothy differedin susceptibility to the pathogens.     

Variation in pea aphid population development in three different habitats

Abstract.

• 1 Seasonal population growth rates for the pea aphid, Acyrthosiphon pisum Harris, were determined in three different host plant habitats; alfalfa, Medicago sativa (L.), clover, Trifolium pratense (L.), and peas, Pisum sativum (L.); over four years and eight places. It was possible to estimate a common intrinsic rate of increase for each host plant habitat.
• 2 An analysis of the relative influence of temporal, spatial and host plant habitat variation showed that the host plant habitat was most important in determining the growth rates of the populations, both in rate of build-up and decline.
• 3 Patterns of alate production in the three different habitats differed substantially between the annual peas and the two perennial legumes. During the summer, alate production was large and rapid in peas and remained low and constant in clover and alfalfa
• 4 Parasitism was highest in peas. The species composition of parasitoids differed between crops.
• 5 Aphids in annual peas had a higher intrinsic rate of increase and a faster rate of decline than in the two perennial legumes. This explains the presence of both migratory and sedentary forms among pea aphids.

     

Abundance and structure of actinomycete complexes in the rhizosphere of winter rye, oats, and red clover

Actinomycete complexes were studied in the rhizosphere of three crop species using luminescence microscopy and plating. The concentration of the total prokaryotic biomass and the length of actinomycete mycelium proved higher in the rhizosphere than in root-free soil. Actinomycetes in the rhizosphere of oats (Avena sativa L.), winter rye (Secale cereale L.), and red clover (Trifolium pratense L.) were represented by the genera Streptomyces and Micromonospora and oligosporous species. The length and biomass of actinomycete mycelium proved to decrease while the generic diversity increased in the following sequence: winter rye—oats—red clover. Increasing soil suppression and plant resistance to phytopathogens using mycelial prokaryotes is discussed in the context of environmental safety.     

Nitrogen transfer from forage legumes to nine neighbouring plants in a multi-species grassland

Legumes play a crucial role in nitrogen supply to grass-legume mixtures for ruminant fodder. To quantify N transfer from legumes to neighbouring plants in multi-species grasslands we established a grass-legume-herb mixture on a loamy-sandy site in Denmark. White clover (Trifolium repens L.), red clover (Trifolium pratense L.) and lucerne (Medicago sativa L.) were leaf-labelled with ¹⁵N enriched urea during one growing season. N transfer to grasses (Lolium perenne L. and xfestulolium), white clover, red clover, lucerne, birdsfoot trefoil (Lotus corniculatus L.), chicory (Cichorium intybus L.), plantain (Plantago lanceolata L.), salad burnet (Sanguisorba minor L.) and caraway (Carum carvi L.) was assessed. Neighbouring plants contained greater amounts of N derived from white clover (4.8?g?m^-2) compared with red clover (2.2?g?m^-2) and lucerne (1.1?g?m^-2). Grasses having fibrous roots received greater amounts of N from legumes than dicotyledonous plants which generally have taproots. Slurry application mainly increased N transfer from legumes to grasses. During the growing season the three legumes transferred approximately 40?kg?N ha^-1 to neighbouring plants. Below-ground N transfer from legumes to neighbouring plants differed among nitrogen donors and nitrogen receivers and may depend on root characteristics and regrowth strategies of plant species in the multi-species grassland.     

Plant Growth Analysis: Allometry, Growth and Interference in Orchardgrass and Timothy

A multiple regression procedure was used to evaluate allometricresponses to stand age and species population densities in monoculturesand mixtures of orchardgrass (Dactylis glomerata L., also knownas cocksfoot) and timothy (Phleum pratense L.). In each speciesthe allometry between shoot dry weight and either leaf areaor tiller number per plant was studied. Population density treatmentsaffected allometry by changing allometric exponents expressingthe ratio of relative growth rates of different plant characteristics.Allometric relationships changed as growth proceeded, and thetwo species differed in their allometric responses to treatments. Plant growth analysis, allometry, competition, Dactylis glomerata L., Phleum pratense L.     

Nitrogen fixation in annual and perennial legume-grass mixtures across a fertility gradient

Background and aims

The selection of legume species and species mixtures influences agroecosystem nitrogen (N) and carbon cycling. We utilized a fertility gradient to investigate the effects of plant species interactions on biological N fixation of an annual and perennial legume in response to shifting soil resource availability.

Methods

Legume N fixation of annual field pea (Pisum sativum) and perennial red clover (Trifolium pratense) grown in monoculture and mixtures with oats (Avena sativa) or orchardgrass (Dactylis glomerata) was estimated using the ¹⁵N natural abundance method across 15 farm fields and we measured six soil N pools ranging from labile to more recalcitrant.

Results

Evidence of complementary and facilitative species interactions was stronger for the perennial red clover-orchardgrass mixture than for the annual field pea-oat mixture (N Land Equivalency Ratios were 1.6 and 1.2, respectively). We estimated that the transfer of fixed N from red clover to orchardgrass increased aboveground N fixation estimates by 15% from 33 to 38?kg?N ha^?1. Despite a more than 2-fold range in soil organic matter levels and more than 3-fold range in labile soil N pools across field sites, the N fertility gradient was not a strong predictor of N fixation. While grass N assimilation was positively correlated with soil N pools, we found only weak, inverse correlations between legume N fixation and soil N availability. In grass-legume mixtures, soil N availability indirectly influenced N fixation through plant competition.

Conclusions

These results suggest that increasing diversity of cropping systems, particularly through the incorporation of perennial mixtures into rotations, could improve overall agroecosystem N cycling efficiency.     

Establishment of reed canary grass with perennial legumes or barley and different fertilization treatments: effects on yield,botanical composition and nitrogen fixation

In two field experiments in northern Sweden, we investigated if intercropping reed canary grass (RCG; Phalaris arundinacea L.) with nitrogen‐fixing perennial legumes could reduce N‐fertilizer requirements and also if RCG ash or sewage sludge could be used as a supplement for mineral P and K. We compared biomass production, N uptake and N‐fixation of RCG in monoculture and mixtures of RCG with alsike clover (Trifolium hybridum L.), red clover (Trifolium pratense L.), goat's rue (Galega orientalis Lam.) and kura clover (Trifolium ambiguum M. Bieb.). In one experiment, RCG was also undersown in barley (Hordeum vulgare L.). Three fertilization treatments were applied: 100 kg N ha^?1, 50 kg N ha^?1 and 50 kg N ha^?1 + RCG ash/sewage sludge. We used a delayed harvest method: cutting the biomass in late autumn, leaving it on the field during the winter and harvesting in spring. The legume biomass of the mixtures at the inland experimental site was small and did not affect RCG growth negatively. At the coastal site, competition from higher amount of clover biomass affected RCG growth and spring yield negatively. N‐fixation in red clover and alsike clover mixtures in the first production year approximately covered half of recommended N‐fertilization rate. Goat's rue and kura clover did not establish well at the costal site, but at the inland site goat's rue formed a small but vital undergrowth. RCG undersown in barley gave lower yield, both in autumn and spring, than the other treatments. The high N treatment gave a higher spring yield at the inland site than the low N treatments, but there were no differences due to fertilization treatments at the coastal site. For spring harvest, there were no yield benefits of RCG/legume intercropping compared with RCG monoculture. However, intercropping might be more beneficial in a two‐harvest system.     

Estimation of nitrogen fixation and transfer from alfalfa to associated grasses in mixed swards under field conditions

Fixation and transfer of nitrogen (N) from alfalfa (Medicago sativa L.) to different grass species including timothy (Phleum pratense L.) and bromegrass (Bromus inermis Leyss) were studied under field conditions, using the¹⁵N dilution technique.The percentage of alfalfa N derived from fixation (%NF) increased throughout the growing seasons and ranged from 62 to 83%. Nitrogen transfer (NT) from alfalfa to associated grasses was evident and contributed 26,46 and 38% of the total annual N yield of associated grasses or represented absolute amounts of 5, 20 and 19 kg N ha^–1 during the first, second and third year, respectively. The gradual and consistent percentage of NT that occurred before first harvest indicated that this transfer is a result of a direct excretion of N compounds from alfalfa root systems. Decomposition of root and nodule debris seems to contribute to the NT from alfalfa to associated grasses in the later cuts. All grass species benefitted similarly from alfalfa, although earlier maturing species with greater competitive ability were slightly more responsive.Contribution No. 1159 from the Plant Research Centre     

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.     

The effects of mixing Italian ryegrass (Lolium multiflorum) with perennial ryegrass (L. perenne) or red clover (Trifolium pratense) on the incidence of viruses

Mixing the ryegrass mosaic virus (RMV) resistant perennial ryegrass (Lolium perenne) cv. Endura with the susceptible Italian ryegrass (L. multiflorum) cv. RvP decreased infection of RvP wth RMV from 37% when grown alone to 22% when mixed. However, Endura yielded less than RvP and there was no yield benefit from mixing the two cultivars. Mixing red clover (Trifolium pratense) cv. Hungaropoly with RvP had no detectable effect on RMV incidence in RvP but did decrease the incidence of red clover necrotic mosaic virus in Hungaropoly from 9% to 1% and of white clover mosaic virus from 53-5% to 41%. The yield of the mixture was equal to that of RvP grown alone but given nitrogen fertiliser. The numbers of eriophyid mites, including Abacarus hystrix the vector of RMV, on ryegrass leaves were similar in pure and mixed swards. It is concluded that with herbage crops, the common practice of sowing mixtures of species may help control virus diseases.     

Nitrogen fixation by white clover when competing with grasses at moderately low temperatures

It was the aim of this study to determine the way in which low temperature modifies the effect of a competing grass on nitrogen fixation of a forage legume. White clover (Trifolium repens L.) was grown in monoculture or in different planting ratios with timothy (Phleum pratense L.) or perennial ryegress (Lolium perenne L.) in growth chambers at either 7.5/5°C (LoT) or 15/10°C (HiT) average day/night temperatures, and with 2.5 or 7.5 mM ¹⁵N-labelled nitrate in the nutrient solution.Competition with grass led to a marked increase in the proportion of clover nitrogen derived from symbiosis (% N_sym). This increase was slower at LoT where % N_sym was reduced considerably; it was closely related to the reduction in the amount of available nitrate as a result of its being utilized by the grass.Nitrogen concentration in white clover herbage and dry matter yield per clover plant were reduced, for the most part, when a competing grass was present. The amount of nitrogen fixed per plant of white clover decreased markedly with temperature. Low temperature consequently accentuated competition for nitrate. The capacity of white clover to compete successfully was limited by its slower growth and nitrogen accumulation.     

Oxygen Supply Limits Nitrogenase Activity of Clover Nodules After Defoliation

The aim of this study was to test the effect of oxygen partialpressure as a possible limiting factor of nitrogen fixationfollowing defoliation. The response of nitrogenase activity(C₂H₂-reduction) of defoliated and undefoliated white and redclover plants (Trifolium repens L. and Trifolium pratense L.)to either 19 kPa oxygen or 55 kPa oxygen was investigated. Priorto defoliation, white clover plants were grown for five weeksin a growth chamber, and red clover plants for 7 or 11 weeksin a glasshouse. The results included measurements of ¹⁶N₂-uptake. Increasing oxygen partial pressure from 19 to 55 kPa severelyrestricted nitrogenase activity of undefoliated white cloverplants; however, 2 h after complete defoliation, the same treatmentcaused a significant increase. A fivefold increase in nitrogenaseactivity upon exposure to the elevated oxygen partial pressurewas found at the end of a 24 h period. This beneficial effectdecreased gradually from 1 to 5 d after defoliation. The responseof recently defoliated red clover plants to 55 kPa oxygen partialpressure was similar to that of white clover, independentlyof plant age. The gradual recovery of nitrogenase activity duringthree weeks of regrowth was associated with a simultaneous changein the response to increased oxygen partial pressure, leadingagain to the response of undefoliated plants. These data suggested that lack of oxygen at the site of nitrogenfixation, resulting from a dramatic increase in oxygen-diffusionresistance, is the main factor limiting nitrogenase activityfollowing defoliation. Trifolium repens L., Trifolium pratense L., white clover, red clover, defoliation, regrowth, nodules, nitrogen fixation, nitrogenase activity, oxygen limitation     

The allelopathic potential of alfalfa root medicagenic acid glycosides and their fate in soil environments

Summary The allelopathic effect of alfalfa (Medicago media Pers.) and red clover (Trifolium pratense L.) root saponins on winter wheat seedling growth and the fate of these chemicals in soil environments were studied. Seed germination, seedling and test fungus growth were suppressed by water and by alcohol extracts of alfalfa roots, and by crude saponins of alfalfa roots, indicating that medicagenic acid glycosides are the inhibitor. Powdered alfalfa roots inhibited wheat seedling growth when added to sand. At concentrations as low as 0.25% (w/w) the root system was completely destroyed whereas seedling shoots suffered little damage. Red clover roots caused some wheat growth inhibition when incorporated to sand, but their effect was much lower than in the alfalfa root treatment. Soil textures had a significant influence on the inhibitory effect of alfalfa roots. The inhibition of seedling growth was more pronounced on light than on heavy soils. This was attribted to the higher sorption of inhibitors by heavy soils. Incubation of alfalfa roots mixed into loose sand, coarse sand, loamy sand and clay loam for a period of 0–8 days resulted in decreased toxicity to bothT. viride and wheat seedlings. This decrease occurred more quickly in heavier soils than in loose sand, due to the hydrolysis of glycosides by soil microorganisms. Soil microbes were capable of detoxifying medicagenic acid glycosides by partial hydrolysis of sugar chain to aglycone. These findings illustrate the importance of medicagenic acid glycosides as an inhibitor of wheat seedling growth, and of their fate in different soil environments.     

The role of boron in plant response to mycorrhizal infection

Summary In a Morrison sandy loam marginal in boron, fertilization with 1.1 ppm boron increased the shoot dry weight of mycorrhizal red clover (Trifolium pratense L.) an average of 16%, but did not affect nonmycorrhizal clover weight. Root colonization and foliar phosphorus concentrations were not significantly affected by B deficiency. With alfalfa (Medicago sativa L.) and Morrison soil in which B deficiency had been intensified by the addition of 100 ppm nitrogen as NH₄NO₃, inadequate B reduced the shoot dry weight of mycorrhizal plants 71%vs a reduction of 35% for nonmycorrhizal plants. Boron deficiency was more severe in the earlier cuttings and delayed the onset of mycorrhizal infection and the subsequent spread of mycorrhizal fungi within the roots. This delay may contribute to the lower concentrations of P and Cu seen by others during early developmental stages of B-deficient alfalfa.     

A Threshold Value for Cutting Intensity in Relation to Fusarium Root Rot of Red Clover (Trifolium pratense L.)

Although cutting the foliage is known to increase Fusarium root rot severity in red clover (Trifolium pratense L.), no quantitative relationship has so far been determined. In this study, results from a number of greenhouse experiments, where plants were artificially inoculated with Fusarium avenaceum (Corda ex Fr.) Sacc., show a linear relationship between cutting intensity and Fusarium root rot in red clover, cv. ‘Hermes II’ an increased cutting intensity giving an increased root rot severity. Theoretically, a threshold value can be calculated beyond which no increase in root rot severity, compared to an uncut plant, should be expected.     

Mapping candidate QTLs related to plant persistency in red clover

Red clover (Trifolium pratense L.) is a diploid (2n = 14), self-incompatible legume that is widely cultivated as a forage legume in cold geographical regions. Because it is a short-lived perennial species, improvement of plant persistency is the most important objective for red clover breeding. To develop a marker-assisted selection (MAS) approach for red clover, we identified candidate QTLs related to plant persistency. Two full-sib mapping populations, 272 × WF1680 and HR × R130, were used for QTL identification. Resistance to Sclerotinia trifoliorum and Fusarium species, as well as to winter hardiness, was investigated in the laboratory and in field experiments in Moscow region (Russia), and Sapporo (Japan). With the genotype data derived from microsatellite and other DNA markers, candidate QTLs were identified by simple interval mapping (SIM), Kruskal–Wallis analysis (KW analysis) and genotype matrix mapping (GMM). A total of 10 and 23 candidate QTL regions for plant persistency were identified in the 272 × WF1680 and the HR × R130 mapping populations, respectively. The QTLs identified by multiple mapping approaches were mapped on linkage group (LG) 3 and LG6. The significant QTL interactions identified by GMM explained the higher phenotypic variation than single effect QTLs. Identification of haplotypes having positive effect QTLs in each parent were first demonstrated in this study for pseudo-testcross mapping populations in plant species using experimental data.