Salt influence on germination and seedling survival of six cool season turfgrass species

Abstract

‘Merion’ Kentucky bluegrass (Poa pratensis L.), ‘Pennfine’ perennial ryegrass (Lolium perenne L.), ‘Seaside’ creeping bent‐grass (Agrostis palustris Huds.), ‘Dawson’ slender creeping red fescue (Festuca rubra trichophylla (L.) Gaud.), ‘Fults’ weeping alkaligrass (Puccinellia distans (L.) Parl.), and ‘common’ Lemmon alkaligrass (Puccinellia lemmoni (Vasey) Scribn.) were evaluated for germination and seedling survival in the greenhouse and laboratory under saline conditions. Overall results indicated that weeping and Lemmon alkaligrass were superior performers under saline and/or sodic conditions. Among the remaining four species, none appeared clearly superior in overall performance.     

Effects of Ozone on Growth of Seven Grass and One Clover Species

Abstract

Seven species of grass and one of red clover were grown at different ozone (O₃) concentrations for about five weeks in growth chambers located in a greenhouse. At an O₃ concentration of 55 compared with 10 nmol mol^?1 during 7 h day^?1 the shoot dry weight decreased by 45% in Phleum pratense, 28% in Dactylis glomerata and Poa pratensis, 23% in Festuca rubra and 16% in Festuca pratensis, while the effects on Agrostis tenuis were not significant. No effect was found at 25 compared with 10 nmol mol^?1O₃. Visible O₃ injury was observed in all six species at 55 nmol mol^?1 and this effect was generally well correlated with the effect on the dry weights. In another experiment no effect was found with O₃ concentrations of up to 62 nmol mol_?1 on Lolium perénne, while the dry weight in Trifolium pratense decreased by 30% at 59 compared with that at 6 nmol mol^?1.     

Silicon Ameliorates the Adverse Effects of Salinity on Turfgrass Growth and Development

An experiment was conducted to assess the effect of foliar application of potassium silicate on Cynodon dactylon [L.] Pers., Festuca arundinacea Schreb. and Lolium perenne L. With increased salinity level, chlorophyll content and relative water content (RWC) in these three turfgrasses were reduced significantly when compared with the untreated control. Supplementary silicon (Si) ameliorated the adverse effects of salinity on chlorophyll content. Silicon treatments decreased proline at all salinity levels. Moreover, addition of Si increased shoot length and shoot number in all turfgrasses. Sodium (Na) concentration was increased in both leaves and roots of turfgrasses at high salinity level; however, Si treatment significantly reduced Na concentration in all of them. Silicon increased Potassium concentration in shoots and roots (though not significantly) in most cases. It can be concluded that Si alleviated the adverse effects of salt stress in all turfgrasses.     

大清河流域河岸植被带污染物净化能力研究

为了解决白洋淀上游河岸带植被类型单一、生态功能低下等问题,从河岸植被带截留、净化面源污染物的生态功能定位出发,选取2种草本和1种灌木为研究对象,通过模拟径流冲刷试验,研究河岸植被带对氮、磷及泥沙污染物的净化效果。结果表明:(1)与单一灌木相比,单一草本的净化效率更好,狗牙根(Cynodon dactylon)的总磷、总氮和泥沙的净化效率最佳,分别为10.88%,6.96%,34.20%;黑麦草(Lolium perenne)对氨氮和硝氮有较好的净化效果,分别为10.11%和9.54%;(2)在多组植物组中,胡枝子(Lespedeza bicolor)、狗牙根、黑麦草的草灌组合净化效率较好,在各项指标平均值上分别较裸土组提高了13.31%,10.84%,9.64%,12.36%,46.43%;(3)灌草和混草的植物组合对5项污染物指标的净化效率均较高,混植组的去除效果优于单一植物组。河岸植被带对面源污染有着一定的净化效果,其中灌草和混草组合的净化效果较好,胡枝子、狗牙根、黑麦草的草灌植物组合表现出最好的净化效果。     

Microsatellite markers and genetic diversity assessment in Lolium temulentum

Lolium and Festuca are two important genera of cool-season forage and turf grasses worldwide. Lolium temulentum L. (darnel ryegrass) has been proposed as a model species for genomics studies of cool-season forage and turf grasses. A study with 41 darnel ryegrass, three tall fescue (Festuca arundinacea Schreb.), two tetraploid fescue (F. glaucescens), and two meadow fescue (F. pratensis) genotypes was initiated to (i) identify a set of microsatellite (simple sequence repeats) markers useful for L. temulentum L., and (ii) to utilize such markers for assessing the genetic variability of L. temulentum accessions collected from different geographical regions of the world. A total of 40 tall fescue (TF) EST-SSRs and 60 Festuca–Lolium (F × L) genomic SSRs were screened on a subset of eight genotypes. The selected 30 tall fescue EST-SSRs and 32 F × L genomic SSRs were used for further analysis of genotypes. The TF-EST- and the F × L genomic-SSRs identified 10.3 and 9.3 alleles per marker, respectively with an average polymorphic information content (PIC) value of 0.66. The phenogram based on 319 EST-SSR and 296 genomic SSR fragments, grouped L. temulentum accessions into three major clusters except for accession ABY-BA 8892.78. Lolium temulentum accession ABY-BA 8892.78 did not cluster with any other accession. The Festuca clusters were distantly related with darnel ryegrass clusters with a similarity coefficient of 0.26. The selected set of tall fescue EST- and F × L genomic SSRs were useful in assessing L. temulentum genetic diversity and could benefit the genetic improvement of members of the Festuca–Lolium complex.     

Comparing cultivars of three cool‐season turf‐grasses for potassium uptake kinetics and potassium recovery in the field

Efficient use of potassium (K) by turf depends on the ability of roots to absorb a high proportion of the fertilizer K applied to the soil. Among turfgrass genotypes, variation in K absorption kinetics of roots and its inheritance is important in the development of genotypes that are more efficient in K absorption from the soil. Therefore, K uptake kinetics of six cultivars each of Kentucky bluegrass (Poa pratensis L.), perennial ryegrass (Lolium perenne L.), and tall fescue (Festuca arundinacea Schreb.) were compared under greenhouse conditions. In 1990 and 1991, field studies of the same cultivars were conducted comparing clipping production rate, leaf blade K concentration, K recovery rate in clippings, K efficiency ratio and visual quality under a moderate K fertilization of 59 kg K/ha/year. Significant differences among species and cultivars were obtained for both absorption kinetics and field recovery of K. Significant correlations between some K uptake parameters and field performance were identified. These results show that genetic differences exist among turfgrasses for K utilization at both the interspecific and intraspecific levels and suggest that a screening program could be developed to identify turfgrass genotypes possessing superior K utilization.     

Use of prolamine polymorphism in studying genetic resources of forage grasses

Summary Electrophoresis of single seed prolamines was used for the analysis ofLolium perenne L.,Festuca pratensis Huds., andDactylis glomerata L. populations. Identification and registration of populations was carried out according to the frequencies of occurrence of genotypes with corresponding types of prolamine banding patterns. The publication sums up the problems of applied use of molecular markers for identification and registration of world genetic resources of forage grasses, analysis of the dynamics of population composition and other problems of plant growing, genetics, breeding and seed control. The approaches mentioned in the article are promising for their use in genetic banks as well as at the institutions which store collections of genetic resources of given crops and at universities and breeding stations.     

Morphological differentiation in four species of the genus Lolium

Three hundred and sixteen accessions belonging to four species of the genus Lolium were analysed for morphological differentiation following field trials. Principal component analysis (PCA) was shown to explain over 74% of the variation with the date of ear emergence, spike height and flag leaf length and width being responsible for the major differentiation. L. perenne separated into two groups following PCA, cluster analysis and k-means clustering, as a result of floral initiation in the sowing year. Those accessions that did not attain floral initiation until the second year were found to be from northern Europe. The inbreeder L. temulentum was separated from the three outbreeding species, L. perenne, L. multiflorum and L. rigidum, which are themselves distinct from each other although there is some introgression between them, especially between L. multiflorum and L. rigidum. The results are discussed in relation to selection of a core collection of Lolium accessions.     

Effect of plant growth-promoting Bacillus sp. on color and clipping yield of three turfgrass species

A two-year irrigated field study was conducted to determine the effects of plant growth-promoting rhizobacteria (PGPR; Bacillus subtilis OSU-142 and Bacillus megaterium M3) as biofertilizer, and in combination with a chemical nitrogen (N) fertilizer, on turf color and clipping yield, and interaction of biofertilizer and chemical N fertilizers in perennial ryegrass (Lolium perenne L.), tall fescue (Festuca arundinacea L. Schreb.), and Kentucky bluegrass (Poa pratensis L.). The three turf species were tested separately in split-plot design experiments with three replications. Three fertilizer sources (ammonium nitrate only, ammonium nitrate + B. megaterium M3, and ammonium nitrate + B. subtilis OSU-142) were the main plots. N applications with monthly applications of 0.0, 2.5, 5.0, and 7.5 g N/m² were the subplots. Color ratings and clipping yields increased with increasing chemical N fertilizers in all species. Both Bacillus sp. significantly increased color ratings and clipping yields in perennial ryegrass and tall fescue. However, there were no significant differences among the three fertilizer sources in color and clipping yield of Kentucky bluegrass. The experiments showed that there is a small but significant benefit from applying biofertilizers for turf color, and that N fertilization may be reduced in some turf species when biofertilization are made for this purpose.     

Quantitative and Qualitative Traits of Natural Ecotypes of Perennial Grasses (Dactylis glomerata L., Festuca arundinacea Schreb., Phalaris tuberosa L., Brachypodium rupestre (Host) R. et S.) Collected in Southern Italy

In 1999, natural populations of Dactylis glomerata L., Festuca arundinacea Schreb., Phalaris tuberosa L. and the macrotherm species Brachypodium rupestre (Host) R. et S., were collected as seed in Southern Italy (Apulia, Basilicata and Campania regions) to evaluate the potential as forage of native germplasm under dry conditions, using available commercial cultivars as controls. The persistence of accessions, biometric, phenologic, productive and qualitative parameters of forage have been studied for a 3-year period (2000–2002) in a typical Mediterranean environment. D. glomerata is widely present in the studied area with two subspecies (subsp. glomerata L., subsp. hispanica (Roth) Nyman). The research pointed out significant differences in plant size, earliness and single plant dry matter (DM) production. All the natural ecotypes belonging to this species showed higher persistence than the control cultivars. Some natural ecotypes appeared to have potential for improving DM yield (subsp. glomerata ecotypes) and reducing neutral detergent fibre (NDF) content (subsp. hispanica ecotypes). Natural ecotypes belonging to F. arundinacea, P. tuberosa, and B. rupestre are sporadically present in the studied area. Among them, two ecotypes of P. tuberosa, showing higher winter growth and earliness compared to cv. ‘Holdfast’, seem to be interesting for a future breeding programme.     

Rhizosphere Influence and Seasonal Impact on Phytostabilisation of Metals—A Field Study

Field experiments were conducted to assess the influence of plant growth and amendment addition on phytostabilisation of copper (Cu), lead (Pb), manganese (Mn) and zinc (Zn) along highway soil in southwest British Columbia, Canada. The plant species tested were Lolium perenne L (perennial rye grass), Festuca rubra L. (creeping red fescue) and Poa pratensis L. (Kentucky blue grass) and the amendments, lime and phosphate. The treatment efficiencies were assessed during different seasons as a completely randomized factorial experiment in split plot design. The research tasks involved: (1) quantifying the seasonal extent of metal accumulation in soil and assessing the seasonal impact on metal speciation for different soil amendments and plant species; (2) determining seasonal accumulation differences between sampling periods in plant parts; and (3) assessing the influence of root–soil interactions on metal dynamics. The amendments decreased the exchangeable fraction and plant uptake of all four metals. The lowest mobile fractions (exchangeable and carbonate bound) were found in soils growing Festuca for Cu, Lolium for Mn and a Lolium/Poa/Festuca combination for Pb and Zn. Metal accumulation and metal dynamics in the rhizosphere soil are compared with those of the bulk soil. The final outcome was the development of a remediation strategy for all four metals involving suitable plants and amendments and incorporating seasonal and rhizosphere influences.     

Effects of arbuscular mycorrhizal fungi on the growth of two turfgrasses grown under greenhouse conditions

ABSTRACT

The need for salinity resistance in turfgrass is increasing because of the enhanced use of effluent and other low-quality water for turfgrass irrigation. Although most turfgrasses form an arbuscular mycorrhizal fungus (AMF) symbiosis, there is little information on the mycorrhization of turfgrass species. Therefore, the aim of this study was to determine the effects of three AMF species, Glomus intraradices Schenck & Smith, Glomus etunicatum Becker & Gerdemann, and Glomus deserticola Trappe & John, and a mixture thereof on the growth, productivity, and nutrient uptake of two species of cool-season turfgrasses, Challenger Kentucky bluegrass (Poa pratensis L.) and Arid tall fescue (Festuca arundinacea Schreb.), and to relate the effects to colonization of the roots by mycorrhiza to assess the dependency of the plants (mycorrhizal dependency [MD]). Following the experimental period (4 months) and measurements, the mycorrhizal inoculated plants had significantly greater biomass production compared to that of non-inoculated plants. MD and shoot mineral contents (particularly P) differed among turfgrass hosting AMF, and the highest value (13%) occurred for P. pratensis and F. arundinacea seedlings colonized with G. intraradices and G. deserticola, respectively. The P content was highest for the F. arundinacea/mixed AMF combination compared to other treatments. We confirmed that mycorrhizal inoculation (P. pratensis/G. intraradices and F. arundinacea/mixed AMF combinations) enhanced plant productivity and nutrient uptake (especially P) even under non-optimum conditions.     

Agronomic evaluations of grass and legume forage species collected in the Xinjiang Autonomous Region of the People's Republic of China and Mongolia

Because rangelands in the Xinjiang Autonomous Region of thePeople's Republic of China (PRC) and Mongolia are typicallyseverely overgrazed, bilateral (U.S./ PRC, U.S./Mongolia)efforts have been initiated to expedite capture of the remaining geneticdiversity. The current study was designed to evaluate selected germplasm of thefollowing forage species collected in these threatened areas: Bromusinermis Leyss., Dactylis glomerata L.,Festuca arundinacea Schreb., Festucaovina L., Festuca rubra L., Phleumphleoides (L.) Karsten, Phleumpratense L., Poa palustris L., Poapratensis ssp. angustifolia (L.) H.Lindb., Medicago sativa ssp. falcataArcengeli, Medicago sativa L. ssp. sativa, Medicago lupulina L.,Medicago ruthenica (L.) Ledebour,Trifolium fragiferum L., Trifoliumlupinaster L., Trifolium pratense L., andTrifolium repens L. Accessions were evaluated in the fieldat Beltsville MD (USA) on an Iuka sandy loam(coarse-loamy, siliceous, acid, thermic, Aquic Udigluvent; pH6.5) in a two-year study. Comparative check cultivars were included.There was significant variation in days to 50% heading (grassspecies) or flowering (legume species), leaf texture (grassspecies), growth habit, spring vigor, and dry matter yield among accessionsof at least some genera. Accessions did not demonstrate higher diseasesusceptibility relative to check cultivars, except in the case of severalF. arundinacea and oneM. sativa ssp.falcata entries. There was no significant variation in leafshape among the legume accessions, nor were leaf shapes significantly differentfrom those of the check cultivars. Several F.ovina, F. rubra,and Poa pratensis accessions may have potential in turfgrass breeding programs. There were apparent positive relationships amongupright growth habit, spring vigor, late development, and yield.     

Characterization of a USDA Kentucky Bluegrass (Poa pratensis L.) Core Collection for Reproductive Mode and DNA Content by Flow Cytometry

Kentucky bluegrass (Poa pratensis L.) is an important turf and forage grass species with a facultative apomictic breeding behavior. In this study, mature seed and leaf tissue from 38 accessions of a USDA core collection of Kentucky bluegrass were analyzed with flow cytometry to characterize the reproductive mode and DNA content for each accession. Major reproductive pathways for each accession were determined based upon the presence and the position of the peaks observed and the known methods of reproduction for Kentucky bluegrass. While the majority of the accessions exhibited facultative apomictic reproductive behavior with a combination of reduced, zygotic and unreduced, parthenogenic embryo production, obligate sexual or obligate apomictic accessions were also found to be present in this core collection. In addition, reduced, parthenogenic and unreduced zygotic embryos were also detected in several accessions. Flow cytometric analysis of somatic tissue revealed a large range of DNA variation within this core collection. We also examined the sensitivity of flow cytometry in analyzing bulked samples containing a large number of plants with varied DNA content and determined that flow cytometry can effectively detect a plant having a different DNA content within a 15-plant bulk sample. Overall the combination of mature seed and somatic tissue analysis generated important information for the Kentucky bluegrass core collection and can be an effective and affordable tool to characterize even greater numbers of Kentucky bluegrass accessions.     

短期NaHCO3胁迫对3种冷季型草坪草生理生态特征的影响

土壤盐渍化严重威胁草坪草的可持续发展,选育和种植耐盐草坪草可改良和利用大面积盐渍土壤。以3种冷季型草坪草黑麦草(Lolium perenne L.)、高羊茅(Festuca arundinacea L.)和早熟禾(Poa pratensis L.)为试验材料,采用盆栽法研究不同浓度NaHCO3胁迫(0,0.2%,0.4%,0.6%,0.8%,1.0%)对3种冷季型草坪草生理生态特征的影响。结果表明:不同浓度NaHCO3胁迫下3种冷季型草坪草草坪外观质量、叶片萎蔫系数、叶片相对含水量、叶片叶绿素含量和K+含量均随着NaHCO3浓度的增加而逐渐降低,且浓度越高,下降越明显;0.4%～1.0%NaHCO3胁迫降低了3种冷季型草坪草的地上部分和根系干重,且随着NaHCO3浓度的增加,生长受到胁迫的抑制程度显著增大,根系部分的受抑制程度比地上部分更明显;不同浓度NaHCO3胁迫下3种冷季型草坪草叶片相对电导率、脯氨酸含量、丙二醛含量和Na+含量随着NaHCO3胁迫浓度的升高呈上升趋势,且浓度越高上升越明显;NaHCO3浓度0.4%时,3种冷季型草坪草已受到伤害;黑麦草、高羊茅和早熟禾在不同浓度NaHCO3胁迫下的隶属函数平均值均表现为早熟禾黑麦草高羊茅,说明3种冷季型草坪草抗NaHCO3胁迫的能力均为早熟禾强于黑麦草和高羊茅。     

Seasonal variations of soil microbial biomass and activity in warm- and cool-season turfgrass systems

Plant growth can be an important factor regulating seasonal variations of soil microbial biomass and activity. We investigated soil microbial biomass, microbial respiration, net N mineralization, and soil enzyme activity in turfgrass systems of three cool-season species (tall fescue, Festuca arundinacea Schreb., Kentucky bluegrass, Poa pratensis L., and creeping bentgrass, Agrostis palustris L.) and three warm-season species (centipedegrass, Eremochloa ophiuroides (Munro.) Hack, zoysiagrass, Zoysia japonica Steud, and bermudagrass, Cynodon dactylon (L.) Pers.). Microbial biomass and respiration were higher in warm- than the cool-season turfgrass systems, but net N mineralization was generally lower in warm-season turfgrass systems. Soil microbial biomass C and N varied seasonally, being lower in September and higher in May and December, independent of turfgrass physiological types. Seasonal variations in microbial respiration, net N mineralization, and cellulase activity were also similar between warm- and cool-season turfgrass systems. The lower microbial biomass and activity in September were associated with lower soil available N, possibly caused by turfgrass competition for this resource. Microbial biomass and activity (i.e., microbial respiration and net N mineralization determined in a laboratory incubation experiment) increased in soil samples collected during late fall and winter when turfgrasses grew slowly and their competition for soil N was weak. These results suggest that N availability rather than climate is the primary determinant of seasonal dynamics of soil microbial biomass and activity in turfgrass systems, located in the humid and warm region.     

Tillage and Forage System Effects on Forage Yields and Nutrient Uptake under Broiler Litter–Amended Soils

Abstract

Planting and harvesting high‐yielding forage grasses may remove phosphorus (P), copper (Cu), and zinc (Zn) from surface soils with a long history of broiler litter application. A study was conducted in Alabama's Sand Mountain region from 1998 to 2000 to determine tillage and forage systems best suited for removing nutrients from such overloaded soils. Tillage treatments included no‐till, moldboard plowing, chisel plowing, and each combined with paraplowing. Forage treatments included bermudagrass (Cynodon dactylon (L.) Pers.) cv. Russell, tall fescue (Festuca arundinacea Schreb.) cv. Kentucky‐31, and an annual rotation of ryegrass (Lolium multiflorum Lam.) and sorghum sudangrass (Sorghum bicolor L. Moench×Sorghum vulgare sudanense). The annual rotation produced highest yields and P uptake. Moldboard plowing the annual rotation further increased yields. It appears the annual rotation best removes P, Cu, and Zn via plant uptake. Tillage reduced P concentrations in the soil surface in the following order: moldboard>chisel>no‐till.     

Potassium uptake of rye-grass (Lolium perenne) and red clover (Trifolium pratense) as related to root parameters

Summary Rye-grass (Lolium perenne) is known to be a strong competitor to red clover (Trifolium pratense) for soil K⁺ under conditions of low K availability in the soil. The objective of this study was to clarify whether this competitive behaviour of the two species can be explained by root morphology. Total K⁺ uptake ofL. perenne andT. pratense was studied under field conditions in relation to root fresh weight, root density, root cation exchange capacity, root surface and root length. The soil was an Alfisol, Udalf. All root parameters, when calculated per unit soil surface (M²), were higher inL. perenne than inT. pratense. In addition,L. perenne had longer root hairs and a denser root hair system thanT. pratense. The greatest difference in root morphology between species was root length, withL. perenne roots averaging 4–6 times longer than those ofT. pratense.Significant correlations were found between the total K⁺ uptake and all root parameters examined, with highest correlationsforroot fresh weight (r,0.92***T. pratense; 0.94***L. perenne) and root length (r, 0.91***T. pratense;r, 0.93***L. perenne). Potassium uptake per unit root fresh weight, root surface and root length were all significantly higher forT. pratense than for L. perenne. Differences in the rate of K⁺ uptake between species were particularly high when expressed per unit root length. Because of its greater root length and surface area,L. perenne can take up more soil K⁺ thanT. pratense, particularly where there is a low K^– supply in the soil. Under such conditionsL. perenne will be a particularly strong competitor toT. pratense.     

修剪留茬高度对北京地区草坪草耗水量的影响

水资源短缺，城市用水紧张，使得草坪灌溉用水管理受到极大关注。为了研究草坪修剪管理的节水效果，采用小型排水式蒸渗仪，通过田间实验研究了充分供水条件下3种草坪草(早熟禾、高羊茅、黑麦)不同修剪高度(留茬高度：5、10、15 cm和不修剪)处理对耗水量的影响，并根据水量平衡原理计算得到了3种草坪草的耗水量。研究结果表明：草坪草的耗水量均随修剪留茬高度增加而增加，不同草种间存在差异。3种草坪草修剪留茬高度为5 cm时较不修剪耗水量减少36.78%(早熟禾)、34.02%(高羊茅)、33.54%(黑麦)，平均减少耗水量34.78%。所得参数可以为类似地区3种草坪草灌溉系统的规划设计和节水管理提供基础理论依据。     

Heavy metal concentration in forage grasses and extractability from some acid mine spoils

Laboratory and greenhouse studies were conducted on several forage grasses, bermudagrass (Cynodon dactylon), creeping red fescue (Festuca rubra), Kentucky 31-tall fescue (Festuca arundinacea), oat (Avena sativa), orchardgrass (Dactylis glomerata), perennial ryegrass (Lolium perenne), sorghum (Sorghum bicolor), triticale (X. triticosecale Wittmack), and winter wheat (Triticum aestivum) grown on three Alabama acid mine spoils to study heavy metal accumulation, dry matter yield and spoil metal extractability by three chemical extractants (Mehlich 1, DTPA, and 0.1 M HCl). Heavy metals removed by these extractants were correlated with their accumulation by several forage grasses. Among the forages tested, creeping red fescue did not survive the stressful conditions of any of the spoils, while orchard grass and Kentucky 31-tall fescue did not grow in Mulberry spoil. Sorghum followed by bermudagrass generally produced the highest dry matter yield. However, the high yielding bermudagrass was most effective in accumulating high tissue levels of Mn and Zn from all spoils (compared to the other grasses) but did not remove Ni. On the average, higher levels of metals were extracted from spoils in the order of 0.1 M HCl>Mehlich 1>DTPA. However, DTPA extracted all the metals from spoils while Mehlich 1 did not extract Pb and 0.1 M HCl did not extract detectable levels of Ni. All of the extractants were quite effective in determining plant available Zn from the spoils. For the other metals, the effective determination of plant availability depended on the crop, the extractant, and the metal in concert.