Evaluation of a cost effective technique for treating aquaculture water discharge using Lolium perenne Lam as a biofilter

Wastewater stabilization ponds generate low cost by-products that are useful for agriculture. The utilization of these by-products for soil amendment and as a source of nutrients for plants requires a high level of sanitation and stabilization of the organic matter, to maintain acceptable levels of soil, water and air quality. In this study, two aquaculture wastewater treatment systems; recirculating system and a floating plant bed system were designed to improve the quality of irrigation water in local communities with low income. In both systems the grass species Lolium perenne Lam was used as a plant biofilter while vegetable specie Amaranthus viridis was used to evaluate the performance of the system and the suitability of the phyto-treated water for irrigation. It was found that the harmful material removal rate for recirculating system was 88.9% for TAN （total ammonia nitrogen）, 90% for NO2--N, 64.8% for NO3--N while for floating plant bed system 82.7% for TAN, 82% for NO2--N and 60.5% for NO3--N. Comparative analysis of the efficiency of waste element removal between the two systems revealed that both systems performed well, however, plant growth was not robust for floating plant bed system while recirculating system is energy consuming. Although both systems did not attain sufficient levels of TN （total nitrogen） and TP （total phosphorus） load reduction, the treatment with L. perenne remarkably improved the irrigation water quality. A. viridis plants irrigated with the phyto-treated discharge water had lesser concentrations of heavy metals in their tissues compared to those irrigated with untreated discharge. The control plants irrigated with untreated discharge were also found to be highly lignified with few stems and small leaves.

Phytoremediation of Heavy Metal Pollution in Urban Sewage River Sediment by Lolium perenne

[Objective] The aim was to restore heavy metal pollution of urban sewage river sediment by using Lolium perenne.[Method] Through cultivation test,the phytoremediation of heavy metal pollution in river sediment by L.perenne was analyzed from the aspects of removal effect,optimal removal time and the changes of sucrase activity,urease activity and microbe quantity.[Result] After planting L.perenne for three months,Ni content in sediment was basically stable,while the removal effect of Cr and Mn in sediment was optimal,and the removal rate of Cr reached the maximum value (57.1%).After 4-5 months,sucrase and urease activity were 4.59 and 10.34 times the previous activity respectively.In addition,the quantity of bacteria and fungi was 2-3 times the previous quantity after 3-4 months.In a word,L.perenne had obvious effects on the removing of heavy metal,improvement of enzyme activity and microbe quantity in polluted river sediment,and the optimal planting time of L.perenne was four months.[Conclusion] The study could provide theoretical foundations for the control of polluted sediment in river.

Transfer DREB into Lolium perenne L. to improve its drought tolerance

A method of Agrobacterium tumefaciens mediated transformation for perennial ryegrass was developed using the calli of ryegrass derived from mature enrbryos. The calli were inoculated with a disarmed A. tumefaciens strain EHA105 harboring binary vector p2328. Vector p2328 contained transcription factor DREB1B and neomycin phosphotransferase （npt H） genes which were driven by promoters of rd29B and CaMV35S, respectively. The inoculated calli were selected on paromomycin- or kanamycin-containing media till the established plants being transferred to soil. Six tmnsgenic plants with DREB1B had been obtained from perennial ryegrass strain Tove. PCR and Southern-blotting showed that npt Ⅱ and DREBIB genes were integrated in perennial ryegrass genome. Stress treatment confirmed that transgenic plants with higher drought tolerance were obtained.

Heterologous expression of Lolium perenne antifreeze protein confers chilling tolerance in tomato

Antifreeze proteins（AFP） are produced by certain plants, animals, fungi and bacteria that enable them to survive upon extremely low temperature. Perennial rye grass, Lolium perenne, was reported to possess AFP which protects them from cold environments. In the present investigation, we isolated AFP gene from L. perenne and expressed it in tomato plants to elucidate its role upon chilling stress. The T1 transgenic tomato lines were selected and subjected to molecular, biochemical and physiological analyses. Stable integration and transcription of Lp AFP in transgenic tomato plants was confirmed by Southern blot hybridization and RT-PCR, respectively. Physiological analyses under chilling conditions showed that the chilling stress induced physiological damage in wild type（WT） plants, while the transgenic plants remained healthy. Total sugar content increased gradually in both WT and transgenic plants throughout the chilling treatment. Interestingly, transgenic plants exhibited remarkable alterations in terms of relative water content（RWC） and electrolyte leakage index（ELI） than those of WT. RWC increased significantly by 3-fold and the electrolyte leakage was reduced by 2.6-fold in transgenic plants comparing with WT. Overall, this report proved that Lp AFP gene confers chilling tolerance in transgenic tomato plants and it could be a potential candidate to extrapolate the chilling tolerance on other chilling-sensitive food crops.

Aquaculture Solids Management Using A Combination of Sand/Gravel or Unwoven Fabric Bed With Lolium perenne Lam as A Plant Biofilter

Objective This work is an evaluation of the efficiency of a sand-gravel or unwoven fabric bed system and Lolium perenne Lam as plant biofilter in the reduction of solids and nutrients removal from aquaculture discharge water. Methods The fzrst step consisted of the collection of wastewater in the tank and the distribution at three different hydraulic loading regimes （0.5, 1, 1.5 L/hour） to the different experimental systems. The second step was to evaluate the performance of the different systems. The fzrst system consisted of a bucket filled with a substrate of sand/gravel （20 cm in depth）, on the bottom of which was a 80 mesh/inch2 of nylon （S1）; the second was similar, but was planted with Lolium perenne lam （S2）; the third was planted with a grass plate consisting of 7 layers of unwoven fabric planted with L. perenne （S3）. Results The second system showed the best performance in reducing solids as well as in nutrients （TN, TP, and COD） reduction. The removal rates for TS, TN, and TP were negatively correlated with the loading regimes, with 0.5 L/hour being the most efficient and thus taken as the reference. Conclusions Solids management using a sand/gravel substrate as bed culture and Lolium perenne L. as plant biofilter has proved to be an efficient technique for solids reduction with low operating cost. This grass plays an important role in wastewater eco-treatment by absorbing dissolved pollutants （TAN） as nutrients for its growth.

Study on Sterilization Method of Endophytic Fungi in Tall Fescue(Festuca arundinacea)and Ryegrass(Lolium perenne)Seeds

[Objectives]This study was conducted to obtain the best solution for sterilizing tall fescue(Festuca arundinacea)and ryegrass(Lolium perenne)seeds by heating in a water bath for a short time.[Methods]The tall fescue and ryegrass seeds infected with endophytic fungi were sterilized by heating in a water bath to compare seed germination and initial growth of turfgrass seedlings under different treatment time.[Results]Sterilization in a 60℃water bath for 20 and 30 min both inhibited the germination of tall fescue and ryegrass seeds,while the 20 min treatment did not significantly affect the lengths of stems,leaves and roots of seedlings.The 20 min water bath sterilization treatment had no effects on the plant heights and biomass of the two turfgrass seedlings and the tiller number of ryegrass,but sterilization for 30 min significantly reduced the aboveground and total biomass of seedlings and the tiller number of ryegrass.[Conclusions]Sterilization in a water bath at 60℃ for 20 min achieved rapid sterilization in a short time,without significantly negatively affecting the growth of seedlings.

Gene Cloning and Expression of the Pyrroline-5-carboxylate Reductase Gene of Perennial Ryegrass(Lolium perenne)

Salt and drought limit the range of applications of perennial ryegrass(Lolium perenne L.), which is one of the important turf and forage grasses. Previous studies have suggested that pyrroline-5-carboxylate reductase(P5CR) might play a central role in proline accumulation in plants that are responsive to stresses. In the present study, the Lolium perenne L. pyrroline-5-carboxylate reductase(LpP 5CR) gene was cloned from leaves of the cultivar ‘Derby' using the RACE technique. The full-length LpP 5CR gene was 1 047 bp in length, which comprised an open reading frame(ORF) of 840 bp in size. Sequence alignment revealed that the putative Lp P5 CR had a 94.3% similarity to Ta P5 CR. q RT-PCR displayed that the mR NA levels of the LpP 5CR gene were almost the same as that in the roots, stems, and leaves of perennial ryegrass seedlings subjected to normal condition or NaC l treatment for 1 h. Moreover, the transcription level of LpP 5CR was up- or down- regulated with Na Cl, polyethylene glycol(PEG), cold, or abscisic acid(ABA) treatment for 3 to 48 h. In addition, confocal microscopy localized the GFP-Lp P5 CR fusion protein to the cytoplasm of onion epidermal cells. These findings suggest that LpP 5CR encodes a cytoplasmic P5 CR protein that plays an important role in the response of perennial ryegrass to various stresses.

Detection of fungal infection in Lolium perenne by Fourier transform infrared spectroscopy

Aims the goal of the study was to apply Fourier transform infrared(FtIr)spectroscopy followed by chemometrical data treatment for the dif-ferentiation of fungi-infected perennial ryegrass(Lolium perenne)from uninfected grass.Methods FtIr was used to rapidly discriminate between leaves of peren-nial ryegrass(L.perenne)infected by a fungal endophyte(Epichloë;asexual forms:Neotyphodium)and uninfected leaves.besides drying and grinding of the sampled leaves,no other preparation steps were needed.FtIr measurements were performed in the attenuated total reflection(at)mode.aliquots of powdered leaf samples were placed on a Znse crystal and the spectra were collected,followed by chemometrical analysis(multidimensional factor analysis,hier-archical cluster analysis).Important Findingsat-FtIr allowed a rapid detection of fungal infections in the plant material and proved to be a fast and reliable tool for the differentiation of plant biomass without the need of time-consuming sample preparation.

Photosynthetic pigments and photosynthetic products of endophyteinfected and endophyte-free Lolium perenne L.under drought stress conditions

Endophyte-infected(EI)seeds of Lolium perenne L.were used to attain endophyte-free(EF)population by heating the seeds at 43℃for 15 min and then 57℃for 25 min.Relative water content(RWC),chlorophyll,soluble sugar and starch content of EI and EF populations under normal and drought stress conditions were compared to investigate the effect of endophyte infection on the host plant.Under severe stress,RWC of EI leaf was significantly higher than that of EF leaf,i.e.EI plants took more advantages over EF plants in water-holding ability.Under mild stress,endophyte could enhance soluble sugars in host plants to improve their osmotic ability.With stress intensification,the improvement of endophyte no longer existed,and more photosynthetic products(such as starch)accumulated in EI plants to survive through the undesirable conditions.In the next spring,EI populations will recover more rapidly than EF populations.The biomass of a population is closely related to its photosynthesis.Under severe stress,EI population significantly accumulated more biomass than EF population.As far as photosynthetic pigments were concerned,contents of Chla,Chlb and Car of EI plants were close to those of EF plants,which suggested that endophyte infection didn’t alleviate photosynthetic pigments from being destroyed by drought stress,and endophyte might improve photosynthesis ability of its host plant in other ways.

Effects of Condensed Tannins from Quebracho Extract on the Kinetic of in vitro Gas Production on Trifofium repens, Lotus corniculatus and Lofium perenne

The aim of this work was to study the effect of condensed tannins （CT） on the kinetic of gas production in vitro, on the three main species of Azorean pasture： Trifolium repens, Lotus corniculatus and Lolium perenne, using Quebracho extract as the source of CT, and also to calculate total tannins and condensed tannins on the species studied. Lotus corniculatus, Lolium perenne and Trifolium repens were tested in vitro with the inclusion of Quebracho extract in doses of 0%, 2.5% and 5% DM. The total content in tannins was determined in the 3 species. We have found, in comparison with standard tannic acid, that tannins exist in the flower of T. repens （0.81 equivalents of tannic acid） and in L .corniculatus （1.07 equivalents of tannic acid） but not in L .perenne （0 equivalents of tannic acid）. The concentration of condensed tannins in the 3 species was： 0.34 mg/mL for T. repens; 0.83 mg/mL for L. corniculatus and 0 mg/ml for L. perenne. We verified that a significant reduction （P 〈 0.05） of gas production happened for doses of 5% of CT in comparison with the other two concentrations： 0% and 2.5% of CT. The results of this study express a reduction of the fermentation rate, which implies a reduction of gas production, so, a reduction of methane emission to the atmosphere and an increase of exploitation of the protein by ruminants.

黑麦草种子及幼苗对镉胁迫的生理响应

为了探明不同黑麦草品种对镉(Cd)富集能力的差异及其生理机制,以6个黑麦草品种为材料,研究镉胁迫对其种子萌发、生物量、镉吸收、色素含量、光合特性及抗逆特性的影响。结果表明,与正常生长条件相比,镉胁迫对不同黑麦草品种种子萌发和根芽生长具有低促高抑作用。镉胁迫增加了黑麦草光合色素含量;镉胁迫增加了维纳斯、奔驰、维多利亚叶片的气孔导度、胞间CO_(2)浓度、蒸腾速率;镉胁迫增加了奔驰、维多利亚、蓝标叶片的POD活性;镉胁迫增加了卡特、维多利亚叶片的SOD活性;镉胁迫增加了维多利亚、维纳斯叶片的CAT活性。奔驰、维多利亚、维纳斯可能较其他品种具有更强的耐镉性,从而减少镉对自身的危害;镉胁迫增加了所有品种可溶性糖含量及除卡特外的5个黑麦草品种脯氨酸含量,奔驰、蓝标、维多利亚能产生较多的渗透调节物质来减轻镉胁迫对其造成的伤害;在正常生长条件下,黑麦草地上部镉含量和镉累积量都较低,品种间差异较小。镉胁迫明显增加了黑麦草地上部镉含量和镉累积量,奔驰地上部镉含量和镉累积量最高,显著高于其他品种。综上,奔驰、维多利亚抗镉性及富镉能力较强,是镉污染土壤修复的优选品种。

黑麦草不同品种响应植被砼干热生境的表型特征

以植被砼生境上的黑麦草(Lolium perenne Linn.)3个品种(Barverdi、Medalist、Accent)为试材,分别进行干热胁迫处理后,测定其伤害比率、叶色、质地、气孔密度和长宽比指标及其恢复能力。结果表明,3个品种的叶色变浅,叶宽减小,伤害表现显著。耐干热性依次为Accent>Medalist>Barverdi,气孔大小为Barverdi>Medalist>Accent,气孔密度为Accent>Medalist>Barverdi。综合评价表明,Accent耐干热性最强。

Study on the Degradation Trend of Ryegrass-White Clover Artificial Grassland

[Objective] The study aimed to explore the degradation law and trend of artificial grassland. [Method] Taking the ryegrass （Lolium perenne） - white clover （ Trifolium repens） artificial grassland in Maiping Township, Guizhou Province as the research object, the grassland vegetation of 40 quadrate from different areas （area around the sheep shed, hilltop, hillside, flatland at the foot of the hill） were analyzed by comparing the dominance and richness index. [ Result] Degradation of different degrees appeared in various areas of this artificial grassland. To be specific, the degradation of grassland around the sheep shed was not serious for less consumption by the sheep; however, the areas on the hilltop and hillside degenerated to the grassland types of dallis grass （ Paspalum dilatatum Poir. ） -Indian lovegrass （ Eragrostis pilosa） and dallis grass - cogongrass [ Imperata cylindrica （ Linn. ） Beauv. ], respectively, and the area at the foot of the hill degenerated to the grassland type dominated by garland chrysanthetnum （ Chrysanthemum coronarium L. ） and knotgrass （ Paspalum distichum L. ）. [ Conclusion ] This study provided a basis for grassland improvement as well as the efficient and sustainable utilization of grazing-type artificial grassland in South China.

Revegetation of Copper Mine Tailings with Ryegrass and Willow

To restore vegetation on metal mine tailings is very difficult because theyoften contain high concentrations of heavy metals, low nutrient content and low water retentioncapacity. This study involved 3 experiments that evaluated the effects of 4 treatment amendments:montmorillonite, rice straw, organic manure and chemical fertilizer on the growth of ryegrass(Lolium perenne L.) and willow (Salix viminalis L.) with Cu and Zn mine tailings from two miningareas. The results showed that ryegrass was the most tolerant of 4 crops to Cu toxicity. Also whenorganic manure, which contained high concentrations of inorganic salts, was added to the minetailings, it significantly hindered ryegrass growth (P = 0.05). Meanwhile, with ryegrass organicmanure significantly increased (P = 0.05) the extractable Cu concentration in both mine tailings.When montmorillonite was used as a mine tailings amendment with willow, the height and tress numberat the 1st cut were significantly greater (P = 0.05) than a control without montmorillonite. Howeverthere was no significant difference for height, tress number, dry weight or root dry weight at the2nd cut. So, amendment applications to reduce metal toxicity and increase nutrients retention inmine tailings were essential during revegetation of mine tailings.

镉胁迫对高羊茅和黑麦草光合特性及叶绿素荧光参数的影响

为探究镉胁迫对高羊茅(Festuca arundinacea)和黑麦草(Lolium perenne)光合特性及叶绿素荧光参数的影响,以高羊茅和黑麦草为试验材料,通过人工盆栽,将Cd^(2+)设置为0(对照)、500、1 000、2 000 mg/kg 4个处理浓度,测定高羊茅和黑麦草的光合特性及叶绿素荧光参数等指标。结果表明,与对照相比,高羊茅的光合作用中净光合速率(Pn)、气孔导度(Gs)、蒸腾速率(Tr)、细胞间CO_(2)浓度(Ci)明显下降,其抑制强度随着Cd^(2+)浓度的升高而加强,但WUE逐渐上升;黑麦草的Pn逐步升高,Gs、Tr、WUE表现为先升后降,Ci明显下降;高羊茅大部分叶绿素荧光参数均明显下降;黑麦草的Fv/Fm显著上升,qP和ETR 2个荧光参数逐渐下降。在镉胁迫下,高羊茅各方面受到的影响都比黑麦草大,因此,在修复被镉污染的土壤中,可以优先选择耐镉能力更强的黑麦草。

基于化感作用的麻风树林下牧草选择性种植研究

以麻风树(Jatropha curcas Linn)叶、外果壳、内果壳混合水浸提液为对象,利用培养皿法及盆栽法对燕麦(Avena sativa L)、黑麦草(Lolium perenne L)和紫花苜蓿(Medicago sativa)3种先锋牧草种子进行萌发试验和生长试验。结果表明,紫花苜蓿种子明显优于燕麦及黑麦草种子,在0.40 mg/mL处理浓度下仍能保持45.67%的发芽率、65.67%的发芽势和51.63%的茎生长抑制率,但燕麦幼苗根生长明显优于紫花苜蓿及黑麦草,在0.40 mg/mL处理浓度时,其根长抑制率为74.47%,显著低于紫花苜蓿的根长抑制率78.16%。

Genome-wide markers for seed yield and disease resistance in perennial ryegrass

The market success of perennial ryegrass(Lolium perenne L.) cultivars depends on sufficient seed production, as they are propagated by seed. However, breeding for high quality forage production reduces seed yield, and breaking the negative correlation would help to overcome the problem. The foliar disease crown rust is another factor affecting reproductive capacity and thereby seed yield. We evaluated seed yield-related traits and resistance to crown rust in a collection of commercial cultivars and ecotypes of perennial ryegrass and identified genome-wide markers associated with the traits. The study revealed high variation between the ecotype and cultivar groups as well as between years. A genome-wide association study identified 17 DNA single-nucleotide polymorphisms(SNPs) of which eight were associated with crown rust and nine with flag-leaf length. The SNP markers were located within or near predicted genes functioning in defense against pathogens. The identified genes are strong candidates for a further in-depth functional study to continue unravel determination of leaf architecture and crown rust resistance in perennial ryegrass.

Forage Species Suitability Mapping for China Using Topographic, Climatic and Soils Spatial Data and Quantitative Plant Tolerances

Selecting plants adapted to the climatic and soil conditions of specific locations is essential for environmental protection and economic sustainability of agricultural and pastoral systems. This is particularly true for countries like China with a diversity of climates and soils and intended uses. Currently, proper species selection is difficult due to the absence of computer-based selection tools. Climate and soil GIS layers, matched with a matrix of plant characteristics through rules describing species tolerances would greatly improve the selection process. Better matching will reduce environmental hazards and economic risks associated with sub-optimal plant selection and performance. GIS-based climate and soil maps have been developed for China. A matrix of quantitative species tolerances has been developed for example forage species and used in combination with an internet map server that allows customized map creation. A web-based decision support system has been developed to provide current information and links to original data sources, supplementary materials, and selection strategies.

Effects of elevated CO_2 levels on root morphological traits and Cd uptakes of two Lolium species under Cd stress

This study was conducted to investigate the combined effects of elevated CO2 levels and cadmium (Cd) on the root morphological traits and Cd accumulation in Lolium multiflorum Lam.and Lolium perenne L.exposed to two CO2 levels (360 and 1000 μl/L) and three Cd levels (0,4,and 16 mg/L) under hydroponic conditions.The results show that elevated levels of CO2 increased shoot biomass more,compared to root biomass,but decreased Cd concentrations in all plant tissues.Cd exposure caused toxicity to both Lolium species,as shown by the restrictions of the root morphological parameters including root length,surface area,volume,and tip numbers.These parameters were significantly higher under elevated levels of CO2 than under ambient CO2,especially for the number of fine roots.The increases in magnitudes of those parameters triggered by elevated levels of CO2 under Cd stress were more than those under non-Cd stress,suggesting an ameliorated Cd stress under elevated levels of CO2.The total Cd uptake per pot,calculated on the basis of biomass,was significantly greater under elevated levels of CO2 than under ambient CO2.Ameliorated Cd toxicity,decreased Cd concentration,and altered root morphological traits in both Lolium species under elevated levels of CO2 may have implications in food safety and phytoremediation.

Stability and purity of selected ryegrass Epichloë endophytes in New Zealand dairy pastures

Background:Perennial ryegrass(Lolium perenne)in New Zealand pastures is typically infected with the mutualist Epichloëfungal endophyte.This endophyte assists the plant in resisting biotic and abiotic stresses,but the standard strain of endophyte is toxic to livestock.Elite ryegrasses with selected endophytes have been developed to provide protective properties to the grass plant and lessen or eliminate the negative impacts on livestock.Methods:Using immunology and molecular techniques,the presence of endophyte infection and endophyte strain in ryegrass tillers was determined for 24 dairy pastures sampled for up to 7 years in regions of the North and South Islands.Results:In general,infection levels were high and showed small increases over time.Some pastures failed to reach 70%infection.The sown,selected endophytes were the dominant endophyte strains present and these were stable over time.Standard endophyte was the primary nonsown endophyte,and while generally low and so of little importance,it increased over time and for some pastures,this would have been detrimental to livestock.Pasture establishment technique influenced the level of contamination.Conclusions:Results reinforce the importance of following best practice procedures in the seed industry and on-farm.Researchers should monitor trials for contaminating nonsown standard endophyte.