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Cross-resistance to ACCase inhibitors of Lolium multiflorum, Lolium perenne and Lolium rigidum found in Chile.

APPs (aryloxyphenoxypropionates) and CHDs (cyclohexanediones) are two of the most important groups used post-emergence for the control of grass weeds. They inhibit the lipid synthesis in plants by interfering with the activity of the enzyme Acetyl-coenzyme A carboxylase (ACCase), acting at a meristematic level. The resistance patterns of the biotypes characterized seem to indicate the existence of different degrees of resistance. It is thus possible to identify biotypes presenting cross-resistance only to certain APPs, to APPs and CHDs, or only to CHDs. The objective of this work was to evaluate the cross-resistance to fenoxaprop-P-ethyl, fluazifop-P-butyl, propaquizafop, cyhalofop-butyl, haloxyfop-R-methyl, tralkoxydim and tepraloxydim in three species of Lolium (L. multiflorum, L. perenne, and L. rigidum) resistant to diclofop-methyl. The assays were conducted with petri-dishes in which, over increasing doses, fifty seeds per biotype and dose were located in each dish. Two weeks later, the following parameters were evaluated: germination (%), number of roots, radicle length, plumule length, and fresh weight reduction (%). Based on plumule length and fresh weight reduction (%), diclofop-methyl resistant biotypes showed cross-resistance to fenoxaprop-P-ethyl, fluazifop-P-butyl, cyhalofop-butyl, haloxyfop-R-methyl, but not to propaquizafop, tralkoxydim and tepraloxydim. The parameters germination (%), number of roots or root length did not show a good relation between the dose and its efficacy (curves of dose response) for any of the susceptible and resistant biotypes studied.

Acetyl-CoA Carboxylase↗

Introgression-mapping of genes for drought resistance transferred from Festuca arundinacea var. glaucescens into Lolium multiflorum.

Procedures for the transfer of genes for drought resistance from Festuca glaucescens (2n=4x=28) into Lolium multiflorum (2n=2x=14) are described. Following the initial hybridisation of a synthetic autotetraploid of L. multiflorum (2n=4x=28) with F. glaucescens, the F1 hybrid was backcrossed twice onto diploid L. multiflorum (2n=2x=14) to produce a diploid Lolium genotype with a single F. glaucescens introgression located distally on the nucleolar organiser region arm of chromosome 3. The transmission of F. glaucescens-derived amplified fragment length polymorphisms and a sequence-tagged-site (STS) marker was monitored throughout the breeding programme. Those genotypes of a mapping population of backcross 3 that survived combined severe drought and heat stress all contained the F. glaucescens-derived markers. The STS marker provided a prototype for a PCR-based system for high-throughput screening during cultivar development for the presence of the F. glaucescens-derived genes for drought resistance. The frequency of intergeneric recombination between L. multiflorum and F. glaucescens is described. During the initial stages of the breeding programme, preferential intraspecific chromosome pairing between Lolium homologues and Festuca homoeologues dominated with low frequencies of intergeneric chromosome associations. However, these increased in the backcross 1 due to the absence of opportunities for intraspecific chromosome pairing between homoeologous Festuca chromosomes following the loss of half of the Festuca chromosomes. Once transferred to Lolium, F. glaucescens sequences recombined with Lolium at high frequencies, thereby enabling the loss of potentially deleterious gene combinations that might reduce the forage quality of Lolium.

Acclimatization↗

Comparison between capillary and polyacrylamide gel electrophoresis for identification of Lolium species and cultivars.

In recent years variety discrimination has been achieved in a range of agricultural crops by means of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Previous works on genus Lolium have shown the effectiveness of this technique for the topic of cultivar identification. In the present research the potential of capillary gel electrophoresis (CGE) for identification of Italian (Lolium multiflorum Lam.), annual (Lolium rigidum Gaud.) and perennial (Lolium perenne L.) ryegrass cultivars in comparison with SDS-PAGE was investigated. Separation conditions of SDS-CGE were chosen in order to obtain electrophoretic data comparable with those of SDS-PAGE, at the expense of analysis speed. Both peak area and migration time of SDS-CGE electropherograms were reproducible. In the examined cultivars, a total of 27 (16.8-96.8 kDa) and 28 (13.2-111.5 kDa) protein subunits were detected by SDS-PAGE and SDS-CGE, respectively. The variability in seed storage protein composition was processed by numerical taxonomy. All cultivars were clearly identified by both electrophoresis systems. The orthogonality between SDS-PAGE and SDS-CGE was suggested by the lack of correlation among protein profiles obtained with the two separation systems. The nonredundant information from these analytical systems should provide a relevant benefit for identification of Lolium cultivars and wild biotypes that are extremely uniform phenotypically.

Electrophoresis, Capillary↗

Androgenesis from Festuca pratensis x Lolium multiflorum amphidiploid cultivars in order to select and stabilize rare gene combinations for grass breeding.

Androgenesis using amphidiploid cultivars of Festuca pratensis x Lolium multiflorum as parents, overcame earlier problems that gave rise to widespread plant sterility amongst androgenic Festulolium populations. Two Festuca pratensis x Lolium multiflorum (2n = 4x = 28) cultivars, Sulino and Felopa, were highly amenable to androgenesis and 10% of plants, including some novel androgenic genotypes, had sufficient fertility to produce progeny and further generations. The genomes of amphidiploid cultivars, which represent the F8 generation, were the result of considerable intergeneric chromosome recombination. Moreover, during cultivar development, natural and breeders' selection pressures had led to the assembly of gene combinations that conferred good growth characters and fertility with the removal of putative deleterious gene combinations. Over 80% of the androgenic plants derived from the amphidiploid F. pratensis x L. multiflorum (2n = 4x = 28) had 14 chromosomes and were likely to be dihaploids with a single genome of Lolium and of Festuca. In contrast, hybrids of F. pratensis x L. multiflorum (2n = 2x = 14) found naturally are invariably sterile. Structural reorganization within the genomes of the androgenic Festulolium plants had restored fertility in genotypes expected to contain the haploid genome of Lolium and Festuca. This provided opportunities for their future incorporation in breeding programmes and the development of fertile diploid Lolium-Festuca hybrids. Amongst the androgenic plants, Festulolium genotypes were recovered that conferred excellent drought resistance or freezing tolerance and were thought to be highly suitable for entry into plant breeding programmes.

Adaptation, Physiological↗

Agrobacterium tumefaciens-mediated transformation of Festuca arundinacea (Schreb.) and Lolium multiflorum (Lam.).

Agrobacterium tumefaciens strain LBA4404 carrying plasmid pTOK233 encoding the hygromycin resistance (hph) and beta-glucuronidase (uidA) genes has been used to transform two agronomic grass species: tall fescue (Festuca arundinacea) and Italian ryegrass (Lolium multiflorum). Embryogenic cell suspension colonies or young embryogenic calli were co-cultured with Agrobacterium in the presence of acetosyringone. Colonies were grown under hygromycin selection with cefotaxime and surviving colonies plated on embryogenesis media. Eight Lolium (six independent lines) and two Festuca plants (independent lines) were regenerated and established in soil. All plants were hygromycin-resistant, but histochemical determination of GUS activity showed that only one Festuca plant and one Lolium plant expressed GUS. Three GUS-negative transgenic L. multiflorum and the two F. arundinacea plants were vernalised and allowed to flower. All three Lolium plants were male- and female-fertile, but the Festuca plants failed to produce seed. Progeny analysis of L. multiflorum showed a 24-68% inheritance of the hph and uidA genes in the three lines with no significant difference between paternal and maternal gene transmission. However, significant differences were noted between the paternal and maternal expression of hygromycin resistance.

Agrobacterium tumefaciens↗

Use of amplified fragment length polymorphism markers to assess genetic diversity of Lolium species from Portugal.

We evaluated the use of amplified fragment length polymorphism (AFLP) markers to distinguish genotypes, populations and species of Lolium. Accessions of two species Lolium perenne and Lolium multiflorum and their hybrid Lolium x hybridum, collected by the Institute of Grass and Environmental Research in 1995 from locations across Portugal, were used. The genetic variation within and between populations from the extremes of latitude and altitude was determined and assessed. Three primer pair combinations generated 765 polymorphic bands. Principal coordinate analysis of similarities between 127 plants showed high dimensionality in the data. Axes 1-3 were associated primarily with species differences, axes 4-14 with population differences within species and axis 15 onwards with within population differences. UPGMA analysis confirmed the groupings. The three populations of L. perenne formed a discrete cluster widely separated from all other populations. There were two distinct groups of L. x hybridum, of which one was similar to and overlapped with L. multiflorum and the second formed a distinct cluster. Analyses of individual bands showed that every inter- and intraspecific contrast involved a different sets of bands, again confirming the high dimensionality of the data. No single band was strictly diagnostic of any population or species. Nevertheless, the UPGMA analysis showed little or no overlap between populations. Thus, despite the high ratio of within-to-between population genetic variance, the full AFLP banding pattern of each genotype is a relatively reliable fingerprint diagnostic of its parent population. The high dimensionality implies that many different factors contribute to the differences observed. This adds to the potential value of the methodology, since it implies that there is a reasonably high likelihood of finding bands relevant to a given environmental gradient or other factor influencing the distribution of genetic diversity.

Cluster Analysis↗

Genomic in situ hybridization (GISH) reveals high chromosome pairing affinity between Lolium perenne and Festuca mairei.

Intergeneric hybridizations have been made between species of Lolium and Festuca. It has been demonstrated, largely through conventional cytogenetic analysis, that the genomes of the two genera are related, however, much information is lacking on exactly how closely related the genomes are between the two species. We applied genomic in situ hybridization (GISH) techniques to the F1 hybrids of tetraploid Festuca mairei with a genomic constitution of M1M1M2M2 and diploid Lolium perenne with a genomic constitution of LL. It was shown in the triploid hybrids (LM1M2) that the chromosomes of M1 and M2 from F. mairei could pair with each other, and it was further discovered that L chromosomes of L. perenne paired with M1 and M2 chromosomes. Our results showed that meiocytes of Lolium-Festuca are amenable to GISH analysis, and provided direct evidence for the hypothesis that the chromosomes of Lolium and Festuca may be genetically equivalent and that reciprocal mixing of the genomes may be possible.

Evolution, Molecular↗

Is Lolium pollen from an urban environment more allergenic than rural pollen?

BACKGROUND: Allergy to grass pollen is a highly prevalent allergic disease. Hay fever is more predominant in urban than in rural areas, despite the increasingly smaller areas of surrounding grassland. The effect of vehicle exhaust pollutants, mainly diesel particles, and other industrial sources of atmospheric pollution leading to plant damage has been implicated in this phenomenon. OBJECTIVE: This study compared the in vivo and in vitro allergenicity of pooled samples of Lolium perenne grass pollen harvested from 10 different urban areas with that of samples of the same pollen from 10 neighboring rural areas. METHODS: Lolium perenne pollen from different parts of a city and from a nearby rural area was harvested in 1999 and 2000 during the peak pollination period. Protein composition was compared by SDS-PAGE and in vivo and in vitro IgE-binding capacity was compared by skin-prick tests, RAST-inhibition and measurement of the major allergen, Lol p 5. RESULTS: In the two years under study, urban samples contained approximately twice the protein content of the rural samples. Biological activity and Lol p 5 content was higher in urban pollen than in rural pollen and showed differences in the two years under study. CONCLUSIONS: The protein content and allergenicity of Lolium perenne pollen was higher in urban areas than in rural areas. These differences might explain why allergy to grass pollen is more prevalent in urban areas. This finding should be taken into account in diagnosis, preventive measures and specific immunotherapy.

Air Pollutants↗

Cold acclimation improves recovery of cryopreserved grass (Zoysia and Lolium sp.).

Cold acclimation of Lolium L. and Zoysia Willd. Grass cultivars significantly increased regrowth of cryopreserved meristems. One wk of cold acclimation improved recovery following cryopreservation but extended acclimation (4-8 wk) resulted in the best regrowth. Cold acclimation also significantly increased the dehydration tolerance of both Zoysia and Lolium meristems. Lolium apices cold acclimated for 4 wk produced 60-100% regrowth following cryopreservation by slow freezing or encapsulation-dehydration. Cold-acclimated Zoysia had greater than 60% regrowth following encapsulation-dehydration when beads were dehydrated to less than 22% water content. Non-acclimated meristems of both genera had little or no regrowth. Thawed meristems grew quickly without callus formation and the plantlets produced were transplanted to pots in the greenhouse after 4 to 6 wk. Samples of each cultivar were stored in liquid nitrogen as part of the U.S. National Plant Germplasm System.

Journal Article↗

The potential of Lolium perenne for revegetation of contaminated soil from a metallurgical site.

A greenhouse study was carried out to determine the possibility of using Lolium perenne for revegetation of soil from a former ferrous metallurgical plant (Naples, South Italy) contaminated by Cu, Pb and Zn at levels above current Italian regulatory limits. Surface soil samples (0-40 cm) from the facility area where raw minerals were disposed (RM1 and RM2), from a nearby unpolluted cultivated soil (C) as control and a 1:3 mixture of the control with the polluted ones (RM1+C and RM2+C) were utilized for the experiment. Revegetation trials were conducted in the greenhouse. At 90 days from seeding, shoot length, chlorophyll content, biomass yield, plant metal uptake and changes of organic carbon content and metal distribution among soil extractable phases defined by sequential extraction were determined. In the mixed substrates (RM1+C and RM2+C) concentrations of Cu, Pb and Zn were still two to three times higher than the Italian regulatory limits. Plants were healthy with 100% survival in all substrates, with no macroscopic symptoms of metal toxicity. The high pH of the soil could be one of the most important parameters responsible for the limited plant availability of the metals. On RM1, RM2 and mixed media, plants experienced retarded growth, reduced shoot length and biomass yield and higher total chlorophyll content compared to those cropped on the control soil, without any evident phytotoxic symptoms. In RM1 and RM2, the plant contents of Cu (19.3 and 12.6 mg kg(-1)), Pb (0.98 and 0.67 mg kg(-1)) and Zn (99 and 88 mg kg(-1)) were higher than that of plants grown on non-contaminated soil (Cu 10.1, Pb < 0.2, Zn 79 mg kg(-1)), but still in the range of physiologically acceptable levels. The distribution of metals in soil was slightly affected by Lolium growth with changes only regarding the organic-bound Cu and Zn pool, with reduction up to 24%. Results indicated that an acceptable healthy vegetative cover can be achieved on the contaminated soil by the proposed revegetation approach and that metals will remain stable over the study period with slight variation of the more available metal forms.

Biomass↗

Nitrogen dynamics under Lolium perenne after a single application of three different sewage sludge types from the same treatment stream.

Three sludge types from the same treatment stream (undigested liquid, anaerobically digested liquid and dewatered, anaerobically digested cake) were used in a field based tub study. Amendments (4, 8, and 16 Mg dry solid (ds)ha(-1)) were incorporated into the upper 15 cm of a sandy loam soil prior to sowing with rye-grass (Lolium perenne L.). Nitrogen transformations in the soil were determined for the 80 d period following incorporation. Nitrogen uptake and crop yield were measured in the cut sward 35 and 70 d after sowing. The study showed that application of sewage sludge at rates as low as 4 Mgha(-1) can have a nutritional benefit to rye-grass over the two harvests. Differences in N transformation, and hence crop nutritional benefit, between sludge types were evident throughout the experiment. In particular, the dewatering process changed the mineral N characteristics of the anaerobically digested sludge, which, when not dewatered, outperformed the other sludges in terms of yield and mineralisation rate at both harvests. The dewatered sludge produced the lowest yield of rye-grass. The undigested liquid sludge had the lowest foliar N and soil NO(3)-N concentrations, possibly immobilised as the large oxidisable C component of this sludge was metabolised by the microbial biomass. Correlation data support the concept of preferential uptake of NH(4)-N over NO(3)-N in Lolium perenne. Results are discussed in the context of managing sludge type and application for a plant nutrient source and NO(3)-N release.

Agriculture↗

Subtribe-specific monoclonal antibodies to Lolium perenne.

BACKGROUND AND OBJECTIVES: The ability to measure personal exposure to airborne grass pollen is important in the understanding of allergic diseases. Visual identification is time consuming and it is difficult to distinguish between many grass pollens morphologically. Although grass pollens share common allergenic determinants, we attempted to produce monoclonal antibodies that would distinguish between species, tribes and subfamilies of grasses which would allow immunodetection of pollens. METHODS: Monoclonal antibodies raised against Lolium perenne were screened for specificity against an extended panel of grass pollen extracts using standard ELISA techniques and a novel particle blotting assay using whole pollen grains. RESULTS: Antibodies showing specificity ranging from subfamily to part-tribe specificity were raised. The most specific monoclonal antibodies (numbers 4, 13 and 17) had reactivity to Lolium perenne and Festuca elatior but displayed little cross-reactivity to Phalaris arundinaceae and the rest of the Poeae tribe when tested by ELISA and no detectable cross-reaction when tested with particle blotting. CONCLUSION: Monoclonal antibodies that are functionally specific to only two grasses can be produced and used to discriminate between related grass species.

Allergens↗

Molecular genecology of temperature response in Lolium perenne: 2. association of AFLP markers with ecogeography.

Improved winter hardiness is an important breeding objective in the forage grass Lolium perenne. This is a complex trait with several components, including the ability to survive and grow at low temperature, to acclimate to cold, tolerate wind, snow cover and ice encasement. Marker-assisted selection has the potential to increase the efficiency of breeding for improved cold tolerance. Here we describe a genecological approach to identifying molecular markers that are associated with adaptation to low winter temperatures. AFLP was used to assess the genetic diversity in 29 wild populations of ryegrass (Lolium perenne) representing a pan-European temperature cline in terms of their geographical origin. A further 18 populations from a temperature cline in Bulgaria were also analysed. In addition, two varieties and five populations representing parents of mapping families currently in use at IGER were included in the analysis. Principal coordinate (PCoA) and cluster analyses of the molecular marker data showed that the Bulgarian altitude cline populations could be distinguished clearly from the other populations. Two regression analyses were carried out; one to identify AFLP markers that correlated in frequency with low mean January temperature of the geographical origin of the population, and another to identify AFLP markers correlating in frequency with the cold tolerance phenotype of the populations, as determined by LT50 values in freezing tests. In the first analysis six AFLP markers showed significant type II trends with mean January temperature, and in the second analysis 28 bands had a significant univariate relationship with the LT50 value of the accessions. In steps 2 and 3 of the stepwise analysis a further 4 and 5 bands, respectively, improved the fit significantly. The results of the two types of regression analysis are discussed in relation to ecogeography and cold tolerance phenotype of the populations.

Ecology↗

Molecular cloning and characterization of a cDNA encoding a ryegrass (Lolium perenne) ENOD40 homologue.

The ENOD40 gene, found in several leguminous and non-leguminous plant species is expressed in the pericycle of legume roots early in the nodulation process, adjacent to the protoxylem poles, but before the cortical cells divide to form the nodule itself. The ENOD40 transcript contains only short open reading frames which give rise to short peptides with a signaling function. The current work reports the cloning and analysis of ENOD40 genes from perennial ryegrass (Lolium perenne) and barley (Hordeum vulgare). Specific polymerase chain reaction (PCR) techniques lead to the isolation of a 659 bp cDNA encoding an ENOD40 homologue, designated LpE-NOD40, from a Lolium perenne stem cDNA library. In addition, a partial ENOD40 cDNA of 384 bp was isolated from barley (Hordeum vulgare) by RT-PCR cloning. The LpENOD40 transcript encodes a putative dodecapeptide, similar to that identified in ENOD40s from leguminous plants and other dicots, and also to ENOD40s from monocots. The coding sequences of ryegrass and barley ENOD40 are represented by a short open reading frame of 12 amino acids. These show a high degree of similarity to each other and to other ENOD40 sequences from monocots. The corresponding genomic DNA from a genomic ryegrass lambda library revealed that the LpENOD40 gene contains no introns. Southern blot analysis shows that the ryegrass genome contains a single copy, possibly two copies, of the gene. Alignment of the ENOD40 cDNA sequences from ryegrass and barley revealed high (77%) nucleotide homology. The ENOD40 peptides are highly conserved, not only among monocots but also on comparison with the dicot peptides. The amino acid identity of region I from ryegrass with its counterparts in maize, barley and rice is 92, 83 and 75% respectively. Expression analysis by RT-PCR demonstrates that a high level of LpENOD40 gene transcript was found expressed in stem tissue, while a lower level was detected in leaves and only a very low expression in flowers of perennial ryegrass. No LpENOD40 transcript was detected in roots.

Amino Acid Sequence↗

A demonstration of a 1:1 correspondence between chiasma frequency and recombination using a Lolium perenne/Festuca pratensis substitution.

A single chromosome of the grass species Festuca pratensis has been introgressed into Lolium perenne to produce a diploid monosomic substitution line 2n = 2x = 14. The chromatin of F. pratensis and L. perenne can be distinguished by genomic in situ hybridization (GISH), and it is therefore possible to visualize the substituted F. pratensis chromosome in the L. perenne background and to study chiasma formation in a single marked bivalent. Recombination occurs freely in the F. pratensis/L. perenne bivalent, and chiasma frequency counts give a predicted map length for this bivalent of 76 cM. The substituted F. pratensis chromosome was also mapped with 104 EcoRI/Tru91 and HindIII/Tru91 amplified fragment length polymorphisms (AFLPs), generating a marker map of 81 cM. This map length is almost identical to the map length of 76 cM predicted from the chiasma frequency data. The work demonstrates a 1:1 correspondence between chiasma frequency and recombination and, in addition, the absence of chromatid interference across the Festuca and Lolium centromeres.

Chromosome Mapping↗

An enhanced molecular marker based genetic map of perennial ryegrass (Lolium perenne) reveals comparative relationships with other Poaceae genomes.

A molecular-marker linkage map has been constructed for perennial ryegrass (Lolium perenne L.) using a one-way pseudo-testcross population based on the mating of a multiple heterozygous individual with a doubled haploid genotype. RFLP, AFLP, isoenzyme, and EST data from four collaborating laboratories within the International Lolium Genome Initiative were combined to produce an integrated genetic map containing 240 loci covering 811 cM on seven linkage groups. The map contained 124 codominant markers, of which 109 were heterologous anchor RFLP probes from wheat, barley, oat, and rice, allowing comparative relationships between perennial ryegrass and other Poaceae species to be inferred. The genetic maps of perennial ryegrass and the Triticeae cereals are highly conserved in terms of synteny and colinearity. This observation was supported by the general agreement of the syntenic relationships between perennial ryegrass, oat, and rice and those between the Triticeae and these species. A lower level of synteny and colinearity was observed between perennial ryegrass and oat compared with the Triticeae, despite the closer taxonomic affinity between these species. It is proposed that the linkage groups of perennial ryegrass be numbered in accordance with these syntenic relationships, to correspond to the homoeologous groups of the Triticeae cereals.

Chromosome Mapping↗

Application of GISH and AFLP techniques for identification of Lolium-Festuca introgressions.

At present, breeding programmes aimed at combining advantageous traits within the Lolium-Festuca complex, are mainly focused on introgression procedures. One principal objective, is the transfer of genes conferring resistance to abiotic stresses from Festuca species (F. pratensis, F. arundinacea and F. glaucescens) into Lolium multiflorum and L. perenne germplasm. In our experiments, two different hybrids: triploid - L. multiflorum (4x) x F. pratensis (2x) and pentaploid - F. arundinacea (6x) x L. multiflorum (4x) were backcrossed twice onto L. multiflorum cultivars, and numerous BC2 progeny generated. BC2 plants from both combinations were tested in field and/or simulated conditions for winter hardiness and drought resistance. GISH (genomic in situ hybridisation) analyses were then performed on the most winter hardy and drought resistant plants to locate putative genes for stress resistance. Using resistant L. multiflorum genotypes with a single Festuca chromatin segment, it was possible to allocate AFLP (amplified fragment length polymorphism) markers specific to that segment. Markers associated with genes conferring stress resistance facilitate marker-assisted selection programmes to obtain new, more persistent grass cultivars. Preliminary results of GISH analysis, to identify Festuca chromosome segments in L. multiflorum introgression lines and to find segment-specific AFLP markers, are presented

Breeding↗

[Establishment and growth of Lolium multiflorum for phyto-remediation of Pb/Zn tailings].

Following the cropping of Sesbania rostrata, Lolium multiflorum was cropped as the subsequent species on an acidified Pb/Zn tailings site. As the experimental treatments, S. rostrata issues harvested on the site were removed from the site or buried into the tailings as the green manure. Changes of tailings chemical and physical properties, germination, growth and heavy metal accumulation of Lolium multiflorum were investigated. The results showed that cropping of S. rostrata improved the chemical and physical properties of the tailings, especially for the contents of organic matter, total nitrogen, available phosphorus and available potassium. The strong acidity (pH < 3) in the Pb/Zn tailings was the main factor that limited the establishment of L. multiflorum. The tailings acidity was decreased temporarily by lime application and it seemed to be helpful only for the germination because the strong acidity was restituted in the end of the experiment. In the most cases, L. multiflorum succeeded to germinate, grow and establish on the Pb/Zn tailings, and yielded relatively great biomass (DM 1.4-3.2 t.km-2). Compared to CK (without S. rostrata cropping), cropping S. rostrata increased the biomass of subsequent L. multiflorum by 4.8%-39.5% in case without S. rostrata green manure using and by 7.7%-139.5% in case with the green manure using, and the green manure application resulted in 2.7%-75.8% increment of L. multiflorum biomass. The results proved that the artificial vegetation process using S. rostrata and subsequent L. multiflorum would be a successful pioneer stage for restoration of the Pb/Zn tailings.

Biomass↗