Grass-legume mixtures sustain strong yield advantage over monocultures under cool maritime growing conditions over a period of 5 years.

Background and Aims: Grassland-based livestock systems in cool maritime regions are commonly dominated by grass monocultures receiving relatively high levels of fertilizer. The current study investigated whether grass-legume mixtures can improve the productivity, resource efficiency and robustness of yield persistence of cultivated grassland under extreme growing conditions over a period of 5 years. Methods: Monocultures and mixtures of two grasses (Phleum pratense and Festuca pratensis) and two legumes (Trifolium pratense and Trifolium repens), one of which was fast establishing and the other temporally persistent, were sown in a field trial. Relative abundance of the four species in the mixtures was systematically varied at sowing. The plots were maintained under three N levels (20, 70 and 220 kg N ha-1 year-1) and harvested twice a year for five consecutive years. Yields of individual species and interactions between all species present were modelled to estimate the species diversity effects. Key Results: Significant positive diversity effects in all individual years and averaged across the 5 years were observed. Across years, the four-species equi-proportional mixture was 71 % (N20: 20 kg N ha-1 year-1) and 51 % (N70: 70 kg N ha-1 year-1) more productive than the average of monocultures, and the highest yielding mixture was 36 % (N20) and 39 % (N70) more productive than the highest yielding monoculture. Importantly, diversity effects were also evident at low relative abundances of either species group, grasses or legumes in the mixture. Mixtures suppressed weeds significantly better than monocultures consistently during the course of the experiment at all N levels. Conclusions: The results show that even in the less productive agricultural systems in the cool maritime regions grass-legume mixtures can contribute substantially and persistently to a more sustainable agriculture. Positive grass-legume interactions suggest that symbiotic N2 fixation is maintained even under these marginal conditions, provided that adapted species and cultivars are used.

Phytotoxic substance with allelopathic activity in Brachiaria decumbens.

The grass Brachiaria decumbens becomes naturalized and quickly dominant in non-native areas. It was hypothesized that phytotoxic substances of plants may contribute to the domination and invasion of the plants. However, no potent phytotoxic substance has been reported in B. decumbens. Therefore, we searched for phytotoxic substances with allelopathic activity in this species. An aqueous methanol extract of B. decumbens inhibited the growth of roots and shoots of cress (Lepidium sativum), lettuce (Lactuca sativa), timothy (Phleum pratense) and ryegrass (Lolium multiflorum) seedlings. The extract was then purified using chromatographic methods and a phytotoxic substance with allelopathic activity was isolated and identified by spectral analysis as (6R,9S)-3-oxo-α-ionol. These results suggest that this compound may contribute to the allelopathic effect caused by the B. decumbens extract and may be in part responsible for the invasion and domination of B. decumbens. Two other Brachiaria species, B. brizantha and a Brachiaria hybrid were also confirmed to contain (6R,9S)-3-oxo-α-ionol. Therefore, this compound may play an important role in the phytotoxicity of the Brachiaria species.

Plant maturity and nitrogen fertilization affected fructan metabolism in harvestable tissues of timothy (Phleum pratense L.).

Timothy (Phleum pratense L.) is an important grass forage used for pasture, hay, and silage in regions with cool and humid growth seasons. One of the factors affecting the nutritive value of this grass is the concentration of non-structural carbohydrates (NSC), mainly represented by fructans. NSC concentration depends on multiple factors, making it hardly predictable. To provide a better understanding of NSC metabolism in timothy, the effects of maturity stage and nitrogen (N) fertilization level on biomass, NSC and N-compound concentrations were investigated in the tissues used for forage (leaf blades and stems surrounded by leaf sheaths) of hydroponically grown plants. Moreover, activities and relative expression level of enzymes involved in fructan metabolism were measured in the same tissues. Forage biomass was not altered by the fertilization level but was strongly modified by the stage of development. It increased from vegetative to heading stages while leaf-to-stem biomass ratio decreased. Total NSC concentration, which was not altered by N fertilization level, increased between heading and anthesis due to an accumulation of fructans in leaf blades. Fructan metabolizing enzyme activities (fructosyltransferase-FT and fructan exohydrolase-FEH) were not or only slightly altered by both maturity stage and N fertilization level. Conversely, the relative transcript levels of genes coding for enzymes involved in fructan metabolism were modified by N supply (PpFT1 and Pp6-FEH1) or maturity stage (PpFT2). The relative transcript level of PpFT1 was the highest in low N plants while that of Pp6-FEH1 was the highest in high N plants. Morevoer, transcript level of PpFT1 was negatively correlated with nitrate concentration while that of PpFT2 was positively correlated with sucrose concentration. This distinct regulation of the two genes coding for 6-sucrose:fructan fructosyltransferase (6-SFT) may allow a fine adequation of C allocation towards fructan synthesis in response to carbon and N availability. Contrary to fructans, starch content increased in low N plants, suggesting different regulatory mechanisms and/or sensitivity of starch and fructan metabolism in relation to the N status.

Effect of lignin content and subunit composition on digestibility in clones of timothy (Phleum pratense L.).

Lignin amount and subunit composition were analyzed from stems and leaf sheaths of timothy (Phleum pratense L.) clones of different in vitro digestibility. Lignin concentration in stems and leaf sheaths was higher in clones of low digestibility than those of high digestibility. No change in lignin concentration occurred in stems as digestibility decreased. Intriguingly, the lignin concentration was lower and the syringyl/guaiacyl (S/G) ratio was higher in stems compared to leaf sheaths at all developmental stages studied. The developmental-associated decrease in digestibility correlated with the increase in S units in lignin in stems and leaf sheaths and in the amounts of p-coumaric acid and ferulic acid residues in the cell wall of stems. Yields of copper oxidation products increased in stems during maturation indicating qualitative changes in the lignin structure. This correlated strongly with the developmentally linked decrease in digestibility. The information obtained is valuable for breeding and for DNA marker development.

Vernalization, gibberellic acid and photo period are important signals of yield formation in timothy (Phleum pratense).

Timothy (Phleum pratense) is a widely grown perennial forage grass in the Nordic region. The canopy consists of three tiller types, of which the stem forming vegetative elongating (ELONG) tiller and generative (GEN) tillers contribute the most to dry matter yield. In this study, the regulation of tiller formation by vernalization, day length (DL) [12 h, short day length (SD); 16 h, long day length (LD)] and gibberellic acid (GA) was investigated in two timothy cultivars. Vernalization resulted in a shift of ELONG to GEN tillers. No vernalization was required for the development of ELONG tillers but SD strictly arrested stem elongation. Vernalization is an important regulator of tiller development but it seemed to be upstream regulated by DL. LD was essential for floral transition and could not be substituted by GA and/or vernalization treatments. Genotypic variation was found in the development of GEN tillers. The ability to produce GEN tillers was associated with significant upregulation of PpVRN3. PpVRN1 expression peaked at the time of vegetative/generative transition, and PpVRN3 after the transfer to LD, suggesting them to have similar functions with cereal vernalization genes. PpVRN1 alone was not sufficient to activate flowering, and upregulation of PpVRN3 possibly together with PpPpd1 was required. Although vernalization downregulated PpMADS10, this gene did not act as a clear flowering repressor. Our results show that flowering signals alter the tiller composition, so they have important effects on yield formation of timothy.

A novel substance with allelopathic activity in Ginkgo biloba.

Ginkgo (Ginkgo biloba) is one of the oldest living tree species and has been widely used in traditional medicine. Leaf extracts of ginkgo, such as the standardized extract EGb761, have become one of the best-selling herbal products. However, no bioactive compound directed at plants has been reported in this species. Therefore, we investigated possible allelopathic activity and searched for allelopathically active substances in ginkgo leaves. An aqueous methanol leaf extract inhibited the growth of roots and shoots of garden cress (Lepidium sativum), lettuce (Lactuca sativa), timothy (Phleum pratense) and ryegrass (Lolium multiflorum) seedlings. The extract was purified by several chromatographic runs and an allelopathically active substance was isolated and identified by spectral analysis to be the novel compound 2-hydroxy-6-(10-hydroxypentadec-11-enyl)benzoic acid. The compound inhibited root and shoot growth of garden cress and timothy at concentrations greater than 3 µM. The activity of the compound was 10- to 52-fold that of nonanoic acid. These results suggest that 2-hydroxy-6-(10-hydroxypentadec-11-enyl)benzoic acid may contribute to the allelopathic effect caused by ginkgo leaf extract. The compound may also have potential as a template for the development of new plant control substances.

Pp6-FEH1 encodes an enzyme for degradation of highly polymerized levan and is transcriptionally induced by defoliation in timothy (Phleum pratense L.).

The ability of grasses to regrow after defoliation by cutting or grazing is a vital factor in their survival and an important trait when they are used as forage crops. In temperate grass species accumulating fructans, defoliation induces the activity of a fructan exohydrolase (FEH) that degrades fructans to serve as a carbon source for regrowth. Here, a cDNA from timothy was cloned, named Pp6-FEH1, that showed similarity to wheat fructan 6-exohydrolase (6-FEH). The recombinant enzyme expressed in Pichia pastoris completely degraded fructans that were composed mainly of ß(2,6)-linked and linear fructans (levan) with a high degree of polymerization (DP) in the crown tissues of timothy. The substrate specificity of Pp6-FEH1 differed from previously characterized enzymes with 6-FEH activity in fructan-accumulating plants: (i) Pp6-FEH1 showed 6-FEH activity against levan (mean DP 20) that was 4-fold higher than against 6-kestotriose (DP 3), indicating that Pp6-FEH1 has a preference for ß(2,6)-linked fructans with high DP; (ii) Pp6-FEH1 had significant activity against ß(2,1)-linked fructans, but considerably less than against ß(2,6)-linked fructans; (iii) Pp6-FEH1 had weak invertase activity, and its 6-FEH activity was inhibited slightly by sucrose. In the stubble of seedlings and in young haplocorms from adult timothy plants, transcripts of Pp6-FEH1 were significantly increased within 3 h of defoliation, followed by an increase in 6-FEH activity and in the degradation of fructans. These results suggest that Pp6-FEH1 plays a role in the degradation of fructans and the mobilization of carbon sources for regrowth after defoliation in timothy.

Vernalization response of Phleum pratense and its relationships to stem lignification and floral transition.

BACKGROUND: Timothy is a long-day grass species well adapted for cultivation in northern latitudes. It produces elongating tillers not only in spring growth but also later in summer. As the quantity and quality of harvested biomass is dictated by canopy architecture and the proportion of stem-forming flowering tillers, the regulation of flowering is of great interest in forage grass production. METHODS: Canopy architecture, stem morphology and freezing tolerance of vernalized timothy were investigated in greenhouse and field experiments. The molecular control of development was examined by analysing the relationship between apex development and expression of timothy homologues of the floral inducer VRN1 and repressor VRN2. KEY RESULTS: True stem formation and lignification of the sclerenchyma ring occur in both vernalized and regrowing stems irrespective of the developmental stage of the apex. The stems had, however, divergent morphology. Vernalization enhanced flowering, and the expression of the VRN1 homologue was elevated when the apex had passed into the reproductive stage. High VRN1 homologue expression was not associated with reduction in freezing tolerance and the expression coincided with increased levels of the floral repressor VRN2 homologue. Field experiments supported the observed linkage between the upregulation of the VRN1 homologue and the transition to the reproductive stage in vernalized tillers. The upregulation of putative VRN1 or VRN2 genes was restricted to vernalized tillers in the spring yield and, thus, not detected in non-vernalized tillers of the second yield; so-called regrowth. CONCLUSIONS: The formation of a lignified sclerenchyma ring that efficiently reduces the digestibility of the stem was not related to apex development but rather to a requirement for mechanical support. The observed good freezing tolerance of reproductive timothy tillers could be one important adaptation mechanism ensuring high yields in northern conditions. Both VRN1 and VRN2 homologues required a vernalization signal for expression so the development of yield-forming tillers in regrowth was regulated independently of the studied genes.

Thresholds, injury, and loss relationships for thrips in Phleum pratense (Poales: Poaceae).

Timothy (Phleum pratense L.) is an important forage crop in many Western U.S. states. Marketing of timothy hay is primarily based on esthetics, and green color is an important attribute. The objective of these studies was to determine a relationship between arthropod populations, yield, and esthetic injury in timothy. Economic injury levels (EILs) and economic thresholds were calculated based on these relationships. Thrips (Thripidae) numbers were manipulated with insecticides in small plot studies in 2006, 2007, and 2008, although tetranychid mite levels were incidentally flared by cyfluthrin in some experiments. Arthropod population densities were determined weekly, and yield and esthetic injury were measured at each harvest. Effects of arthropods on timothy were assessed using multilinear regression. Producers were also surveyed to relate economic loss from leaf color to the injury ratings for use in establishing EILs. Thrips population levels were significantly related to yield loss in only one of nine experiments. Thrips population levels were significantly related to injury once before the first annual harvest and twice before the second. Thrips were the most important pest in these experiments, and they were more often related to esthetic injury rather than yield loss. EILs and economic thresholds for thrips population levels were established using esthetic injury data. These results document the first example of a significant relationship between arthropod pest population levels and economic yield and quality losses in timothy.

Specific bottom-up effects of arbuscular mycorrhizal fungi across a plant-herbivore-parasitoid system.

The majority of plants are involved in symbioses with arbuscular mycorrhizal fungi (AMF), and these associations are known to have a strong influence on the performance of both plants and insect herbivores. Little is known about the impact of AMF on complex trophic chains, although such effects are conceivable. In a greenhouse study we examined the effects of two AMF species, Glomus intraradices and G. mosseae on trophic interactions between the grass Phleum pratense, the aphid Rhopalosiphum padi, and the parasitic wasp Aphidius rhopalosiphi. Inoculation with AMF in our study system generally enhanced plant biomass (+5.2%) and decreased aphid population growth (-47%), but there were no fungal species-specific effects. When plants were infested with G. intraradices, the rate of parasitism in aphids increased by 140% relative to the G. mosseae and control treatment. When plants were associated with AMF, the developmental time of the parasitoids decreased by 4.3% and weight at eclosion increased by 23.8%. There were no clear effects of AMF on the concentration of nitrogen and phosphorus in plant foliage. Our study demonstrates that the effects of AMF go beyond a simple amelioration of the plants' nutritional status and involve rather more complex species-specific cascading effects of AMF in the food chain that have a strong impact not only on the performance of plants but also on higher trophic levels, such as herbivores and parasitoids.

Improvement of plant growth and nickel uptake by nickel resistant-plant-growth promoting bacteria.

In this study, among a collection of Ni-resistant bacterial strains isolated from the rhizosphere of Alyssum serpyllifolium and Phleum phleoides grown on serpentine soil, five plant growth-promoting bacteria (PGPB) were selected based on their ability to utilize 1-aminocyclopropane-1-carboxylate (ACC) as the sole N source and promote seedling growth. All of the strains tested positive for indole-3-acetic acid (IAA) production and phosphate solubilization. In addition, four of the strains exhibited significant levels of siderophores production. Further, the efficiency of PGPB in enhancing Ni solubilization in soils was analyzed. Compared with control treatment, inoculation of PGPB strains significantly increased the concentrations of bioavailable Ni. Furthermore, a pot experiment was conducted to elucidate the effects of inoculating Ni-resistant PGPB on the plant growth and the uptake of Ni by Brassica juncea and B. oxyrrhina in soil contaminated with 450 mg kg(-1) Ni. Psychrobacter sp. SRA2 significantly increased the fresh (351%) and dry biomass (285%) of the B. juncea test plants (p<0.05), whereas Psychrobacter sp. SRA1 and Bacillus cereus SRA10 significantly increased the accumulation of Ni in the root and shoot tissues of B. juncea compared with non-inoculated controls. This result indicates that the strains SRA1 and SRA10 facilitated the release of Ni from the non-soluble phases in the soil, thus enhancing the availability of Ni to plants. A significant increase, greater than that of the control, was also noted for growth parameters of the B. oxyrrhina test plants when the seeds were treated with strain SRA2. This effect can be attributed to the utilization of ACC, solubilization of phosphate and production of IAA. The results of the study revealed that the inoculation of Ni mobilizing strains Psychrobacter sp. SRA1 and B. cereus SRA10 increases the efficiency of phytoextraction directly by enhancing the metal accumulation in plant tissues and the efficient PGPB, Psychrobacter sp. SRA2 increases indirectly by promoting the growth of B. juncea and B. oxyrrhina.

Timothy hay with a low dietary cation-anion difference improves calcium homeostasis in periparturient Holstein cows.

The current study was undertaken to evaluate the effects of feeding timothy (Phleum pratense L.) hay differing in dietary cation-anion difference (DCAD) on the capability of cows to maintain calcium homeostasis around parturition. We hypothesized that feeding low-DCAD timothy hay during the prepartum period would induce a mild metabolic acidosis prepartum and improve calcium homeostasis postpartum with no effect on dry matter intake. Forty-one dry pregnant Holstein cows entering their second lactation or greater were used in a randomized complete block design. Timothy hay was obtained from an established timothy stand under a pivot irrigation system. Low-DCAD timothy hay was produced by fertilizing the area between the second and third pivot towers at a rate of 224 kg of CaCl(2)/ha, and control timothy hay (high DCAD) was grown on the area between the fourth and fifth pivot towers of the same field. The chloride concentration was 1.07 and 0.15% on a dry matter (DM) basis, and the DCAD was 1.2 and 21.6 mEq/100 g of DM for the low- and high-DCAD timothy hay, respectively. Experimental diets, containing timothy hay at 63% of dietary DM, were fed ad libitum starting 30 d before the expected calving date. The DCAD values were 1.6 vs. 14.5 mEq/100 g of DM for the low- and high-DCAD timothy-based diets, respectively. At the beginning of the study, urine pH and blood bicarbonate concentration averaged 8.22 +/- 0.06 and 28.5 +/- 0.3 mM, respectively. The low-DCAD timothy diet decreased urine pH compared with the high-DCAD timothy diet on d 21 (7.75 vs. 8.31), d 14 (7.69 vs. 8.22), and d 7 (7.50 vs. 8.19) before calving, and it also decreased the prepartum blood bicarbonate concentration by 2 mM. In addition, cows fed the low-DCAD timothy diet had greater blood ionized calcium concentration prepartum (1.22 vs. 1.19 mM), greater blood ionized calcium concentration at 0 and 8 h after calving, and similar prepartum dry matter intake. These results indicate that timothy hay differing in DCAD affects the acid-base balance of periparturient dairy cows, and that low-DCAD timothy hay improves calcium homeostasis postpartum with no negative effect on dry matter intake.

Compost, manure, and gypsum application to timothy/red clover forage.

Some of the most fertile agricultural land in Atlantic Canada includes dykelands, which were developed from rich salt marshes along the Bay of Fundy through the construction of dykes. A 2-yr field experiment was conducted on dykeland soil to evaluate the effect of fertility treatments: source-separated municipal solid waste (SS-MSW) compost, solid manure, commercial fertilizer, and gypsum on (1) timothy/red clover forage productivity, (2) N, S, and other nutrients uptake, and (3) residual NO(3)-N and NH(4)-N in the soil profile. All fertility treatments increased dry matter yields from the two cuts each year relative to the control. Residual soil NO(3)-N and NH(4)-N concentrations in the fall of the second year decreased with depth, and beyond 20-cm depth were lower than 1 mg kg(-1). Gypsum application equivalent to 40 kg S ha(-1) increased dry matter yields and N uptake by forage, and increased soil Mehlich 3-extractable S, tissue S, and uptake of S, Ca, P, Cu, Fe, and Mn relative to the control. High rates of compost can provide sufficient N, S, and perhaps other nutrients to a perennial forage system under the cool wet climate of Atlantic Canada with no heavy metal enrichment of forage. However, the chemical N provided greater total N uptake than organic sources, except the high rate of compost, suggesting that the N availability from organic sources was not well synchronized with forage N demand. Municipal solid waste compost may also increase soil and forage tissue Na, which might be of concern.

Quantification of photoperiodic effects on growth of Phleum pratense.

BACKGROUND AND AIMS: Accurate quantifications of plant responses to photoperiod are useful for physiological studies, in growth modelling and in other studies of environmental effects. The objective of the current work was a mathematical description of photoperiodic influence on plant morphological traits, using functions with few and common parameters related to key plant characteristics and typical response patterns. METHODS: Two latitudinal cultivars of timothy (Phleum pratense) were studied in a climate chamber experiment at 9, 12, 15, 18, 21 and 24 h photoperiods. Seedling growth was recorded by measurements of main tiller leaf tip heights every other day from the 5-6 leaf stage onwards, and as plant size and dry weight at days 37, 46, 62 and 70, i.e. at the end of experiment. The plant responses to photoperiod were described by the term PPR = (eci(PP-PPc))/(1 + e(ci+di)(PP-PPc)), where PP = photoperiod in h, PPc = photoperiod of maximum response, c = characteristic coefficient of main response interval, d = sensitivity coefficient characterizing course of function beyond the main response interval. PPR was tested on experimental data for different growth characteristics (i), e.g. size of individual leaves (Yi), identified by their sequential numbers on the main tiller (LN) using the function: Yi = Ybi + aiLN + biLNalphai (PRR). The growth course was described by the same function, replacing LN with day number of treatment exposure. KEY RESULTS AND CONCLUSIONS: The functions described with high precision (r2 > 0.97) the effect of photoperiod on growth as expressed by several plant characteristics, such as leaf area development, top and root DM production, as well as cultivar differences. Green leaf area was more sensitive to photoperiod than above-ground DM production. The southern cultivar 'Grindstad' was more sensitive than the northern one 'Engmo'. The functional relationships suggest mechanisms for plants' daylength responses and latitudinal adaptation.

Nitrogen availability from composts for humid region perennial grass and legume-grass forage production.

Perennial forages may be ideally suited for fertilization with slow N release amendments such as composts, but difficulties in predicting N supply from composts have limited their routine use in forage production. A field study was conducted to compare the yield and protein content of a binary legume-grass forage mixture and a grass monocrop cut twice annually, when fertilized with diverse composts. In all three years from 1998-2000, timothy (Phleum pratense L.)-red clover (Trifolium pratense L.) and timothy swards were fertilized with ammonium nitrate (AN) at up to 150 and 300 kg N ha(-1) yr(-1), respectively. Organic amendments, applied at up to 600 kg N ha(-1) yr(-1) in the first two years only, included composts derived from crop residue (CSC), dairy manure (DMC), or sewage sludge (SSLC), plus liquid dairy manure (DM). Treatments DM at 150 kg N ha(-1) yr(-1) and CSC at 600 kg N ha(-1) yr(-1) produced cumulative timothy yields matching those obtained for inorganic fertilizer. Apparent nitrogen recovery (ANR) ranged from 0.65% (SSLC) to 15.1% (DMC) for composts, compared with 29.4% (DM) and 36.5% (AN). The legume component (approximately 30%) of the binary mixture acted as an effective "N buffer" maintaining forage yield and protein content consistently higher, and within a narrower range, across all treatments. Integrating compost utilization into livestock systems that use legume-grass mixtures may reduce the risk of large excesses or deficits of N, moderate against potential losses in crop yield and quality, and by accommodating lower application rates of composts, reduce soil P and K accumulation.