Hide and seek – Infection rates and alkaloid concentrations of Epichloë festucae var. lolii in Lolium perenne along a land‐use gradient in Germany

The common forage grass Lolium perenne has evolved with the systemic fungal endophyte Epichloë festucae var. lolii. The endophyte provides herbivore resistance to the grass due to defensive alkaloids, some of which are toxic to grazing livestock. In this field study, we determine whether distribution of the endophyte‐grass association changes along a land‐use intensity gradient on 87 managed grasslands in three German regions. Endophyte infections were detected in 66% of the studied sites and infection rates within infected sites ranged from 1% to 95%. Alkaloid concentrations of lolitrem B (vertebrate toxin) exceeded the toxicity thresholds in 50 (14%) of 351 infected plants and of peramine (invertebrate deterrent/toxin) in 12 (3%) of 351 plants. Infection rates and alkaloid concentrations were not significantly affected by land‐use intensity and region, but alkaloid concentrations were higher in summer compared to spring. We conclude that risks for livestock intoxication are currently low, as (i) average alkaloid concentrations per grassland were always below toxicity thresholds and as (ii) none of the grasslands was dominated by L. perenne. We suggest avoidance of grass monocultures in Europe to keep intoxication risks for livestock low; we also recommend regular examination of seeds and grasslands, as seed producers might accidentally distribute infected seeds, and as climate warming might further enhance the distribution of Epichloë endophytes in European grasslands.     

The infection of Festuca rubra subsp. pruinosa by Epichloë festucae

Festuca rubra subsp. pruinosa is a grass that inhabits the sea cliffs of the Atlantic coasts of Europe. Four populations of this plant in the northern coast of Galicia, in northern Spain, were analysed for the presence of fungal endophytes. Of the plants included in the study, on average 0·69 were infected by an intercellular fungal endophyte. Using morphological and molecular characters, this endophyte was identified as Epichloë festucae, a clavicipitaceous, seed‐transmitted species. A half‐sibling line, consisting of endophyte‐free and endophyte‐infected plants, was developed and used to test whether the presence of the fungus affected the response of the plants to salinity. Dry weights of infected or uninfected plants, watered with sea‐water solutions or tap water, did not significantly differ.     

Fungal alkaloids in populations of endophyte-infected Festuca rubra subsp. pruinosa

Festuca rubra subsp. pruinosa is a grass that grows on coastal cliffs along the Atlantic coast of Europe. Asymptomatic plants of this species are systemically infected by the fungal endophyte Epichloë festucae. It is not known whether the alkaloids, ergovaline and peramine, are produced by the endophyte in F. rubra subsp. pruinosa. Plants from four populations were collected from the northern coast of Galicia (Spain) and examined for the presence of fungal endophytes. Ergovaline and peramine concentrations were analysed over two consecutive years, at two plant growth stages, and in different types of plant tissues. Infected plants of F. rubra subsp. pruinosa contained ergovaline but not peramine. Ergovaline was detected in 0·80 of the plants, with concentrations ranging from 0·05 to 1·9 μg g^?1 dry matter. The differences in ergovaline concentration between different types of plant tissues (vegetative and reproductive), plant populations and sampling dates were not statistically significant.     

Fungal endophytes in grasses from semi-arid permanent grasslands of western Spain

Abstract A survey of grass species associated with Epichloë and Neotyphodium fungal endophytes was made in permanent semi‐arid grasslands of western Spain. Endophytes were isolated from 11 of 49 grass species analysed. Infected plants of Festuca arundinacea, Festuca rubra, Dactylis glomerata and Lolium perenne were detected at most locations studied. The grasses, Agrostis castellana, Brachypodium phoenicoides and Festuca ampla, are previously unknown endophyte hosts. The results obtained show that endophyte–grass interactions are present in the grasslands where this study was conducted.     

Identification,ecological evaluation and phylogenetic analysis of non‐symbiotic endophytic fungi colonizing timothy grass and perennial ryegrass grown in adjacent plots

Non‐systemic endophytes coexist with grasses and produce positive or negative effects for the host. In agricultural grasses, endophytes such as Epichloë spp. (formerly: Neotyphodium spp.) enhance the biometric parameters and agricultural value of grass biomass and seeds. Some endophytic fungi produce active substances that exert a negative influence on grass‐fed livestock. There is a general scarcity of studies investigating other endophytic fungi, the species composition of fungal communities, fungal species capable of colonizing different grasses and endophyte transfer between grass taxa. This study aims to fill in the existing knowledge gap by describing the relationships between fungal species and grass species. Timothy grass (Phleum pratense L.) is more readily colonized by endophytic fungi than perennial ryegrass (Lolium perenne L.), and the ratio of fungi isolated from the above species was determined at 3:1. Ecological indicators, especially diversity, were also higher in the fungal community colonizing timothy grass. The vast majority of the isolated fungi were ascomycetes. In addition, two Basidiomycota isolates and three Oomycota isolates (Phythium acanthicum) were also obtained from timothy grass. The most prevalent fungal species were Alternaria alternata, Microdochium bolleyi and Epicoccum nigrum. An analysis of minisatellite DNA regions revealed high levels of genetic polymorphism in A. alternata, whereas the remaining isolates were characterized by low levels of genetic variation or genetic homogeneity. The transfer of endophytic fungi between grass species was determined, which was one of the most important observations made in the study. The Sørensen–Dice coefficient reached 50%, which indicates that all fungal species isolated from perennial ryegrass are capable of colonizing timothy grass.     

Beneficial effects of Neotyphodium tembladerae and Neotyphodium pampeanum on a wild forage grass

Asexual, vertically transmitted fungal endophytes of the genus Neotyphodium are considered to enhance growth, stress resistance and competitiveness of agronomic grasses, but have been suggested to have neutral or deleterious effects on wild grasses. We studied whether the associations between Bromus auleticus and their endophytes, Neotyphodium pampeanum and Neotyphodium tembladerae, are beneficial for this wild forage grass native to South America. In a greenhouse experiment, 3‐month‐old endophyte‐infected plants (E+) showed enhanced growth relative to their endophyte‐free counterparts (E?). In a one‐year‐long experiment in the field, E+ plants showed higher survival and regrowth rate, and produced more biomass and seeds than E? plants. Only with respect to the seed output did N. tembladerae seem to be detrimental, whereas N. pampeanum did not affect this parameter. No differences were observed with respect to the germination of seeds produced by E+ or E? plants. Loline alkaloids were detected in N. pampeanum‐infected plants. Our results show that, similar to results with agronomic grasses, in wild grasses, the symbiosis with Neotyphodium species could be mutualistic.     

Influence of boron on seed yield and seed quality of organic white clover (Trifolium repens L.)

The effects of boron (B) application on flower development, nectar production, germination and seed yield were investigated in organic seed crops of white clover grown in soils with <1 mg B kg^?1. Application techniques of a liquid B product were studied in two field experiments in 2009–2011. Three rates of B application to the soil at sowing and three rates of B as a foliar application in the harvest year were compared with untreated control plots. None of the B treatments had a significant effect on seed yield, relative to the control. In one experiment, with a dense crop stand, the results indicated a small (10%) yield increase with B applied to the soil. Seed germination and B concentration of shoots, roots and seeds were unaffected by B application, although the soil concentration increased. In seeds with an average B concentration of 22 mg kg^?1, 73% germinated as normal seedlings and 14% were assessed as hard seeds; corresponding values for seeds with an average B concentration of 17 mg kg^?1 were 60 and 38% respectively. The results indicate the need for further studies of factors determining B uptake in white clover .     

Effect of ascorbic acid on seed germination of three halophytic grass species under saline conditions

Grasses on the Pakistani coast are moderately to highly salt tolerant and have potential for utilization as a cash crop. This study was designed to determine whether seed germination of three halophytic grasses (Phragmites karka, Dichanthium annulatum and Eragrostis ciliaris) could be improved by exogenous application of ascorbic acid (AsA) under saline conditions. Seeds of P. karka were germinated in varying concentrations of NaCl and AsA under different temperature regimes, and seeds of Dichanthium annulatum and Eragrostis ciliaris were germinated at optimal temperatures only. In P. karka, concentrations of AsA (5 and 10 mM) alleviated the salinity effects better at cooler and moderate thermo‐periods, whereas higher concentrations (20 mM of AsA) failed to improve germination under all temperature regimes. AsA was ineffective at a warmer thermo‐period (25/35°C). The rate of germination also increased at all thermo‐periods with the application of AsA except at 25/35°C under saline conditions. Application of AsA improved the germination of E. ciliaris seeds under saline conditions but was inhibitory for D. annulatum in comparison with the untreated control. The rate of germination followed the similar pattern as that of seed germination. Results indicate that AsA has the ability to partially alleviate the effect of salinity on seed germination of some grass species under optimal temperature regime.     

‘MaxClover’ grazing experiment: I. Annual yields,botanical composition and growth rates of six dryland pastures over nine years

Six dryland pastures were established at Lincoln University, Canterbury, New Zealand, in February 2002. Production and persistence of cocksfoot pastures established with subterranean, balansa, white or Caucasian clovers, and a perennial ryegrass‐white clover control and a lucerne monoculture were monitored for nine years. Total annual dry‐matter (10.0–18·5 t DM ha^?1) and sown legume yields from the lucerne monoculture exceeded those from the grass‐based pastures in all but one year. The lowest lucerne yield (10 t ha^?1 yr^?1) occurred in Year 4, when spring snow caused ungrazed lucerne to lodge and senesce. Cocksfoot with subterranean clover was the most productive grass‐based pasture. Yields were 8·7–13·0 t DM ha^?1 annually. Subterranean clover yields were 2·4–3·7 t ha^?1 in six of the nine years which represented 26–32% of total annual production. In all cocksfoot‐based pastures, the contribution of sown pasture components decreased at a rate equivalent to 3·3 ± 0·05% per year (R² = 0·83) and sown components accounted for 65% of total yield in Year 9. In contrast, sown components represented only 13% of total yield in the ryegrass‐white clover pastures in Year 9, and their contribution declined at 10·1 ± 0·9% per year (R² = 0·94). By Year 9, 79% of the 6.6 t ha^?1 produced from the ryegrass‐white clover pasture was from unsown species and 7% was dead material. For maximum production and persistence, dryland farmers on 450–780 mm yr^?1 rainfall should grow lucerne or cocksfoot‐subterranean clover pastures in preference to ryegrass and white clover. Inclusion of white clover as a secondary legume component to sub clover would offer opportunities to respond to unpredictable summer rainfall after sub clover has set seed.     

Seed characterization and direct sowing of native grass species as a management tool

There is insufficient information about germination, seed biology and seedling establishment of native grass species of the cerrado (Brazilian savannah) to recommend their use in restoration of disturbed areas. We evaluated the production, percentage of fertile seeds, germination, seedling growth and establishment in the field of eighteen native grass species and one exotic invasive species (Melinis minutiflora) and also the effects of different storage periods on germination. The percentage of fertile seeds varied from 6·0 to 94·2% among native species. Ten species produced a minimum of 800 fertile seeds per gram. Germination rate was in the range of 11·0 to 98·8%. Storage for 1 year reduced the germination rate in one species, did not affect germination of three species and increased germination (by 2–10 times) in 14 species. After 2 years of storage, six species showed reductions in germination. Three different patterns of resource allocation were observed and might provide mechanisms for increasing seedling establishment under different environmental conditions. In the field, only two species flowered in the first year after sowing and an additional eight in the second year of observation. Melinis minutiflora is an aggressive species with more than three times the fertile seed production of native species (2820 seeds per gram), high germination rate (55%) and a high number of germinable seeds (1551 per gram). Despite the wide variation in seed characteristics and the delay in flowering, it is concluded that most native grass species can be used in projects aiming at the restoration and management of degraded areas.     

Occurrence of endophytes in grasses native to northern China

A survey of grass species associated with Neotyphodium and Epichloë fungal endophytes was made in the permanent grasslands of northern China. A total of 41 grass species, 56 sites and 172 local populations were examined to reveal endophytic infection of native grasses. Twenty‐five of the forty‐one species of grasses (proportionately 0·61) were infected by endophytes: twenty‐two species had a mean infection rate of 0·01–0·50 and only three species had a mean infection >0·50. Of the 172 local populations examined, however, proportionately 0·72 were not infected, 0·181 had an infection rate of 0·01–0·50 and 0·099 had an infection rate of 0·51–1·00. Two genera of grasses, Cleistogenes and Koeleria, as well as twenty plant species, were previously unknown endophytic hosts in China. High infection rates (0·86–1·00) of plants of Achnatherum sibiricum were detected at all eleven sites studied. Other grasses in which high infection rates were found were Agropyron mongolicum, Roegneria turczaninovii, Agropyron elongate, Poa palustris and Poaangustifolia. It was concluded that endophytes are found widely in natural grass populations and that endophyte–grass interactions may have effects on ecosystems and livestock in the grasslands of northern China.     

Mice find endophyte‐infected seed of tall fescue unpalatable – implications for the aviation industry

Bird strike is a significant problem for the aviation industry, caused in part by the attractiveness of the grass surrounding airports to birds. Endophyte‐infected grasses such as Avanex^® have been shown to reduce bird populations at airports through the production of secondary metabolites. These metabolites are unpalatable to herbivorous, omnivorous and granivorous birds and are known to reduce insect densities, thereby making the sown areas less attractive to insectivorous birds. Raptors also provide a bird‐strike threat which could be reduced by controlling mouse populations around airports. In this study, the effect of endophyte‐infected seed on mouse feeding behaviour has been investigated. By performing a choice trial offering diets containing endophyte‐infected and endophyte‐free tall fescue seed, it was shown that endophyte‐infected seed was less palatable to mice (P < 0·001 in a 14‐d trial). Furthermore, when diet positions were reversed, mouse feeding behaviour was modified to again select endophyte‐free material (P < 0·001 over a further 14 d). This result shows that endophyte‐infected grasses could not only be used in the control of bird populations but they also have the potential to control mouse populations which in turn would decrease the number of raptors. This use of endophyte‐infected grasses in areas surrounding airport runways shows great promise for the aviation industry.     

Seed growth of three perennial ryegrass cultivars sown on two dates and treated with trinexapac ethyl straw shortener

First‐year crops of diploid perennial ryegrass (cvs. Meridian, Bronsyn and Grasslands Impact) were sown on 1 April and 14 May 2008. Applications of trinexapac ethyl (TE) plant growth regulator at 0, 200 and 400 g a.i. ha^?1 were used to shorten stems to examine the impact of seed growth. Seed filling followed a consistent sigmoidal growth pattern with a lag phase of 127°C days, and linear duration of 390°C days. Time to 95% of final seed weight was 517°C days. Seed yield increases from TE were from higher numbers of first‐grade seeds m^?2, achieved by a higher rate of seed filling during the linear phase of 0·115 mg per °C day per spike. For all cultivars, the maximum stem dry weight occurred at 310–400°C days post‐anthesis, which suggest the stem was a strong sink. As seeds developed, their demand for assimilate increased and they drew more from the stem. At harvest, stem weights from TE treatments were 25% heavier than at anthesis, while untreated ‘Bronsyn’ and ‘Grasslands Impact’ stems were similar to those at anthesis. Thus, stems treated with TE contributed assimilates to increase seed yield but were still a net sink with assimilates in the stem at harvest. Trinexapac ethyl rate induced an inverse relationship between seed yield and stem height. This showed that competition for assimilate between stems and growing seeds limited the seed yield. Management or genetic factors that reduce stem height are likely to increase seed yields of perennial ryegrass.     

Effects of additives on the fermentation and aerobic stability of grass silages and total mixed rations

Mown herbage of timothy–meadow fescue (dry matter 218 (LDM) or 539 (HDM) g kg^?1) was ensiled in laboratory silos to evaluate silage additives. For LDM silage, additives including formic acid (a blend of formic acid, sodium formate, propionic acid, benzoic acid, glycerol and another blend of formic acid and ammonium formate, both applied at 5 L t^?1) were able to restrict fermentation and thereby improve intake potential of the silage. Aerobic stability (AS) of total mixed ration (TMR) was also improved. LDM grass treated with homofermentative lactic acid bacteria (_hoLAB) resulted in silage containing lactic acid at 132 g kg^?1 DM, ammonium‐N <40 g kg^?1 total N, and pH < 3·8, and the AS was poor (<36 h). The treatment including heterofermentative strain (Lactobacillus brevis) produced more acetic acid and better AS than _hoLAB. Salt treatment (sodium benzoate, potassium sorbate, sodium nitrite) reduced pH compared to the Control treatment (3·89 vs. 4·24) and improved AS of TMR. The LDM Control silage had good AS, but the TMR based on it had poor AS. All additives were able to lower pH on HDM silages also, but other benefits of using additives were minimal. The treatment including L. brevis on HDM was able to improve AS of TMR.     

Germination responses of the halophyte Chloris virgata to temperature and reduced water potential caused by salinity,alkalinity and drought stress

Chloris virgata is considered a useful grass species for grassland restoration in northern China. However, little information exists concerning the germination responses of this species to temperature and water potential caused by stress conditions. Experiments were conducted in growth chambers to assess the effect of temperature, salinity, alkalinity, drought and the interactions of temperature and stress on seed germination. Seeds were germinated at three diurnal temperature regimes, with four water potentials in NaCl, NaHCO₃ and PEG solutions. Results showed that optimal germination under stress occurred at 15–25 °C, and germination percentages and rates were inhibited by either an increase or decrease in temperature from the optimal temperature. The inhibitory effects of the low water potential caused by salinity and drought on germination were greater at 25–35 °C, but seeds were subjected to more stress despite the relatively higher water potential because of the alkalinity at this temperature. The recovery percentage under salinity was highest at ?1·2 MPa at 15–25 °C, and more than 80% of seeds also germinated at this water potential after they were transferred from drought stress. However, seeds lost their viability in higher alkalinities under all temperatures, and at 25–35 °C, there was lower recovery percentage under stress. Results suggest that salinity, alkalinity and drought stress have different impacts on seed germination, and the tolerance to stress of C. virgata seeds is affected by the interactions of temperature and water potential caused by salinity, alkalinity and drought. Chloris virgata shows potential utility as a promising grass species in salinity–alkalinity and drought‐stressed environments.     

Lactic acid bacteria strains for enhancing the fermentation quality and aerobic stability of Leymus chinensis silage

Leymus chinensis is an important grass in China and Russia. Six lactic acid bacteria (LAB) strains (LB, LPL1, LPL2, LPL3, LCL and WH) from L. chinensis silage were screened and identified and their effects on fermentation quality were investigated. All six strains were grown at 6·5% NaCl and pH 4·00. Strains LPL1, LPL2 and LPL3 were identified as Lactobacillus plantarum, and LB, WH and LCL were classified as Lactobacillus brevis, Weissella hellenica and Lactobacillus casei respectively. The six isolated strains and a commercial inoculant (Lactobacillus buchneri) were added to L. chinensis for ensiling at densities of 500 and 600 kg m^?3. The control was sprayed with the same volume of distilled water. The effects of the strains on fermentation quality after 45 d ensiling and aerobic stability during 8 d of exposure to air were evaluated. The 600 kg m^?3 silage had lower pH, butyric acid, ammonia nitrogen content and coliform bacteria counts than the 500 kg m^?3 density silage (P < 0·05). The six isolated strains decreased pH, butyric acid content and increased lactic acid content, and all inoculants increased L. chinensis silage aerobic stability except LCL (P < 0·05). The fermentation quality of L. chinensis silage increased with higher ensiling density. The LAB strains improved the fermentation quality, and high‐quality silage could be obtained at low ensiling density with the addition of the LAB strains. The strains improved the aerobic stability; Lb. buchneri and Lb. brevis showed the best performance.     

Species interactions in a grassland mixture under low nitrogen fertilization and two cutting frequencies: 1. dry‐matter yield and dynamics of species composition

Four‐species mixtures and pure stands of perennial ryegrass, tall fescue, white clover and red clover were grown in three‐cut and five‐cut systems at Ås, southern Norway, at a low fertilization rate (100 kg N ha^?1 year^?1). Over a three‐year experiment, we found strong positive effects of species diversity on annual dry‐matter yield and yield stability under both cutting frequencies. The overyielding in mixtures relative to pure stands was highest in the five‐cut system and in the second year. Among the possible pairwise species interaction effects contributing to the diversity effect, the grass–grass interaction was the strongest, being significant in both cutting systems and in all years. The grass–legume interactions were sometimes significant, but no significant legume–legume interaction could be detected. Competitive relationships between species varied from year to year and also between cutting systems. Estimations based on species identity effects and pair‐specific interactions suggested that the optimal proportions of red clover, white clover, perennial ryegrass and tall fescue in seed mixtures would have been around 0·1, 0·2, 0·4 and 0·3 in the three‐cut system, and 0·1, 0·3, 0·3 and 0·3 in the five‐cut system.     

Seed dormancy and dormancy‐breaking methods in Leymus chinensis (Trin.) Tzvel. (Poaceae)

Leymus chinensis (Trin.) Tzvel. is a perennial grass with high productivity and forage value; however, poor stand establishment, often due to seed dormancy, limits its widespread use for forage production. To investigate the mechanism of seed dormancy and to develop effective methods of improving germination, the contribution of each part of the caryopsis to dormancy was investigated, and a number of single or combined dormancy‐breaking pre‐treatments were conducted using three seed lots. The palea, lemma, pericarp/testa, and endosperm all contributed to seed dormancy. The contribution of each part to dormancy was 23·4%, lemma; 6·2%, palea; 28·4%, pericarp/testa; and 42·0%, endosperm. Hull (palea and lemma) removal, pericarp/testa piercing, and soaking in distilled water or 30% sodium hydroxide (NaOH) significantly decreased the percentage of dormant seeds (i.e. increased germination). Treating hull‐removed and pericarp/testa‐pierced seeds with gibberellic acid (GA₃) also significantly decreased the percentage of dormant seeds. Compared with each of the single pre‐treatments, the combined pre‐treatment of pre‐soaking in water for 1 d, then 30% NaOH for 60 min and treating with 300 μm GA₃ resulted in the highest germination (89%); and seed viability was 91%.     

Species interactions in a grassland mixture under low nitrogen fertilization and two cutting frequencies. II. Nutritional quality

Mixtures and pure stands of perennial ryegrass, tall fescue, white clover and red clover were grown in a three‐cut and a five‐cut system in southern Norway, at a low fertilization rate (100 kg N ha^?1 year^?1). The nutritional quality (annual weighted averages) of the dried forage from the two‐first harvesting years was analysed. There was no significant effect of species diversity on crude protein (CP) concentration. In the three‐cut system, we found a significant species diversity effect leading to 10% higher concentrations of acid detergent fibre (ADF), 20–22% lower concentrations of water‐soluble carbohydrate (WSC) and 4% lower net energy for lactation (NE_L) concentrations in mixtures compared with pure stands (averaged across the two‐first years). In the five‐cut system, similar effects were seen in the first year only. This diversity effect was associated with a reduction in WSC and NE_L concentrations and an increase in ADF, NDF and CP concentrations in the grass species, and not in red clover, when grown in mixtures. This is thought to be a combined result of better N availability and more shading in the mixtures. Species diversity reduced the intra‐annual variability in nutritional quality in both cutting systems.     

三种浸种剂对Na2SO4胁迫下小麦种子萌发期耐盐性的影响

为了解黄腐酸、褪黑素、水杨酸三种浸种剂对硫酸盐胁迫下小麦种子萌发期耐盐性的调节效应,分别采用不同浓度的黄腐酸(0.5、1.0和1.5 g·L^-1)、褪黑素(0.01、0.05和0.10 mmol·L^-1)、水杨酸(0.1、0.5和1.0 mmol·L^-1)对小麦进行浸种处理,随后与未处理的小麦种子一并采用100 mmol·L^-1 Na₂SO₄溶液培养,测定各处理下小麦种子萌发期的生长生理指标,并通过主成分分析综合评价其耐盐性。结果表明,0.5～1.5 g·L^-1的黄腐酸、0.01～0.10 mmol·L^-1褪黑素、0.1～0.5 mmol·L^-1水杨酸浸种均提升了小麦根系活力、体内抗氧化酶活性,降低了小麦体内超氧阴离子自由基■产生速率及丙二醛(MDA)含量,缓解了盐分对小麦种子萌发的胁迫程度,增强了小麦种子萌发期的耐盐性,促进了小麦的萌发生长。其中,1.5 g·L^-1的...