See discussions, stats, and author profiles for this publication at: /publication/294577448 Effect of heavy metals on ed germination and edling growth of common ragweed and roadside ground cover legumes
Article in Environmental Pollution · June 2016
DOI: 10.vpol.2015.11.041
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Effect of heavy metals on ed germination and edling growth of common ragweed and roadside ground cover legumes
Jichul Bae a,Diane L.Benoit b,Alan K.Watson a,*
a Department of Plant Science,McGill University(Macdonald Campus),21111Lakeshore Roads,Ste.Anne de Bellevue,Qu e bec H9X3V9,Canada
b Saint-Jean-sur-Richelieu Rearch and Development Centre,Agriculture and Agri-Food Canada,430Gouin Boulevard,Saint-Jean-sur-Richelieu,Qu e be
c J3B3E6,Canada
a r t i c l e i n f o
Article history:
Received13May2015 Received in revid form
11November2015
Accepted24November2015 Available online xxx
Keywords:
Ambrosia artemisiifolia
Heavy metal
Supplement ground cover Germination
Seedling growth
Seedling mortality a b s t r a c t
In southern Qu e bec,supplement roadside ground Trifolium spp.)struggle to establish near edges of major roads and thus fail to assist turf recruitment.It creates empty niches vulnerable to weed establishment such as common ragweed(Ambrosia artemisiifolia).We hypothesized that heavy metal stress may drive such species shifts along roadside edges.A growth chamber experiment was con-ducted to asss effects of metals(Zn,Pb,Ni,Cu,and Cd)on germination and edling behaviors of roadside weed(A.artemisiifolia)and ground cover legumes(Coronilla varia,Lotus corniculatus,and Trifolium arven).All metals inhibited T.arven germination,but the effect was least on A.artemisiifolia. Low levels of Pb and Ni promoted germination initiation of A.artemisiifolia.Germination iculatus was not affected by Zn,Pb,and Ni,but inhibited by Cu and Cd.Germination of C.varia was decread by Ni,Cu,and Cd and delayed by Zn and Pb.Metal additions hindered edling growth of all test species,and the inhibitory effect on the belowground gr
owth was greater than on the aboveground growth.Seedling mortality was lowest in A.artemisiifolia but highest in T.arven when expod to the metal treatments.L. corniculatus and C.varia edlings survived when subjected to high levels of Zn,Pb,and Cd.In conclusion, the successful establishment of A.artemisiifolia along roadside edges can be associated with its greater tolerance of heavy metals.Thefindings also revealed iculatus is a potential candidate for supplement ground cover in metal-contaminated roadside edges in southern Qu e bec,especially sites contaminated with Zn and Pb.
©2015Elvier Ltd.All rights rerved.
1.Introduction
Roadsides along major roads are a hostile environment for turf-grass establishment(Brown and Gorres,2011).To help roadside turf establishment,certain species of perennial legumes are commonly utilized as supplemental ground cover(NCDOT,1998;Sincik and Acikgoz,2007).Roadsides along primary roads and highways in southern Qu e bec,Canada have been planted with perennial le-gumes,usually clover(Trifolium spp.)cultivars,together with a cold-ason salt-tolerant turf grass mix(GTCVC,2006).However, the clovers often struggle to establish near pavement edges and fail to
support permanent turf-recruitment.This results in creating vacant niches vulnerable to weed establishment along roadside edges.Ambrosia artemisiifolia(common ragweed)has frequently exploited the empty niches and colonized along roadside edges in southern Qu e bec(DiTommaso,2004).Roadside mowing does not control A.artemisiifolia as mowed plants develop condary branches below cutting height(Simard and Benoit,2011).
A.artemisiifolia is not only a noxious agricultural weed,but also one of the most prevalent allergenic weeds.In Qu e bec, A.artemisiifolia pollens cau asonal rhino-conjunctivitis and dermatitis,which affects1in7people(over million people)in the province(MSSS,2012).Up to10%of the overall US population is nsitive to ragweed pollen(Wilken et al.,2002).Additionally,the species is spreading across many parts of Europe and Asia,where it pos threats to public health and native ecosystems(Bullock et al., 2010;Fang and Wan,2009).Roadways contributed to the spread of A.artemisiifolia in both native and invaded regions(Brandes and Nitzsche,2006;Joly et al.,2011;Lavoie et al.,2007;Milakovic et al.,2014;Vitalos and Karrer,2009).Therefore,management of roadside populations of A.artemisiifolia is necessary to prevent its further spread.
*Corresponding author.
E-mail address:alan.watson@mcgill.ca(A.K.
Watson).
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Environmental Pollution213(2016)112e118
野山谷Soil heavy metal concentrations in the vicinity of major roads in the metropolitan area of Montr e al,Q
u e bec often exceed environ-mental thresholds(Cloutier-Hurteau et al.,2014;Ge et al.,2000). Surface soils(pH7.5e7.8)of roadside edges along heavy-traffic highways(!30,000vehicles per day)in southern Qu e bec contain Zn,Pb,and Cu at bioavailable levels phytotoxic to nsitive species (Bae et al.,2015).Metals generally cau a concentration-dependent inhibition of ed germination and edling growth, but the degree of inhibition varies depending on plant species and metal types(Kranner and Colville,2011).A respon to metal stress during the plant's early life cycle can be one of the potential drivers underlying establishment success near heavy-traffic roadside edges.We found that A.artemisiifolia emergence was positively correlated with Zn,Pb and Cu bioavailability,while Trifolium arven(rabbitfoot clover)emergence was negatively correlated (Bae et al.,2015).We hypothesized that A.artemisiifolia establish-ment along roadside edges may ari from its tolerance to metal stress,which provides a competitive advantage over the current roadside cover Trifolium spp.).Therefore,a growth chamber experiment was conducted to evaluate ed germination and edling growth of A.artemisiifolia(common ragweed),T. arven(rabbitfoot clover),Lotus corniculatus(birdsfoot trefoil)and Coronilla varia(crown vetch)in respon to heavy metals(Zn,Pb, Ni,Cu and Cd).
2.Materials and methods
不攻自破的意思
2.1.Seed collection and storage
In November2011,mature plants(13e15plants per site)of A.artemisiifolia were randomly collected from six roadside sites along Qu e bec Route104in Saint-Jean-sur-Richelieu(45.19 N, 73.16 W),Qu e bec,Canada.They were dried for one week at room temperature and then eds were extracted.Seeds iculatus,C.varia and T.arven were purchad from Richters (Goodwood,ON),Picked Eastern Canada(Saint-Hyacinthe,QC), and Indigo(Ulverton,QC)in December2011.All eds were stored in the dark at4 C in a cold storage room with40%relative humidity.
2.2.Metal treatments
Zinc(Zn),lead(Pb),nickel(Ni),copper(Cu),and cadmium(Cd) were ud becau they are the most common metals encountered in roadside topsoil in southern Qu e bec(Cloutier-Hurteau et al., 2014).For germination trials,Zn,Pb,Ni,and Cu treatments were 0,50,100,and200mg/kg and Cd treatments were0,5,and10mg/ kg.For edling growth tests,Zn,Pb,Ni and Cu treatments were0, 25,50,75,and100mg/kg and Cd treatments were0,2.5,5,7.5,and 10mg/kg.The concentrations were bad on the background metal levels in soils of southern Qu e bec(50mg Pb/kg;100mg Zn/kg; 50m
g Ni/kg;50mg Cu/kg;and1.5mg Cd/kg)(MDEFP,2003).Metal solutions were prepared by diluting10,000mg/kg(Zn,Pb,Ni,and Cu)and1000mg/kg(Cd)of ICP standard solution of each metal (99.9%,SCP Science,Canada)with deionized water.The control was deionized water.
2.3.Seed germination experiment
2.3.1.Seed preparation
Six hundred eds of A.artemisiifolia and C.varia were soaked in 6%sodium hypochloride solution for5min and rind by running deionized water for10min.The eds were placed on two layers of filter paper(Whatman No.1)moistened with5ml of deionized water in ten petri dishes(60eds per dish)and the dishes were stored in the dark at4 C for six weeks.Six hundred eds iculatus and T.arven were scarified by120-grit sandpaper and wet-cold stratified(60eds per dish)for24h.
2.3.2.Germination substrate and condition帐篷哥
Metal treatment solutions were solidified with0.8%(w/v)Bacto agar(DIFCO)and adjusted to pH5.8prior to autoclaving becau metal phytotoxicity asssment on agar media is more nsitive thanfilter papers(Di Salvatore et al.,2008).The metals were applied as ICP standard metal solutions
dissolved in4%nitric acid. Since agar solidification is pH dependent(an optimal range be-tween5.4and5.8)and most metal ions usually begin to precipitate above pH6,potassium hydroxide(2M KOH)was added to adjust to pH5.8.We found no effect of the itric acid,potas-sium hydroxide,nitrate,and potassium)on germination of the test species(Bae et al.,2014).
Micro centrifuge tubes(1.5ml)were ud to avoid statistical issues in the petri-dish germination test(Bae et al.,2014).A small hole was made in the tube cap to allow airflow.After autoclaving and cooling at room temperature,1.3ml of treatment media was added to each micro centrifuge tube.The stratified eds were examined under a discting microscope and pressure-tested with a forceps.Badly deteriorated eds and tho that did not resist the slight pressure were discarded.For each treatment,25tubes with each test species were assigned as a replicate and each replicate was randomly assigned on tube racks.The tubes were transferred to a growth chamber with alternating temperatures of10 C(10h dark cycle)and25 C(14h light cycle),60%relative humidity and 6100lu/m2light intensity.The alternating temperatures and pho-toperiods were similar to thefield conditions during late spring or early summer in southern Qu e bec.Due to the chamber capacity, each germination trial was conducted for each metal type with three replicates for each treatment and was repeated one more time.
2.3.3.Germination experiment
写给她的歌
The number of germinated eds was recorded every24h for two weeks.A ed was considered to have germinated when an emerging radicle was longer than2mm.Tubes with germinated ed were removed.Germination respon variables were germi-nation rate(T50)andfinal germination percentage(TG).Germina-tion rate(T50)referred to the time(hours)to reach50%offinal germination over the two-week trial and was calculated by the following formula(Farooq et al.,2005):
T50¼
&
N
2
蒸发糕的做法Àn i
'À
t iÀt j
Á
À
n iÀn j
Á
where N is thefinal number of ed germination at two weeks and ni,nj cumulative number of eds germinated by concutive counts at times ti and tj measured in hours when ni<N/2<nj.Final germination percentage(TG)at the end of the experiment period was calculated by the following formula:
TG¼
Number of germinated eds at2weeks
Total number of eds
Â100
2.4.Seedling growth experiment
2.4.1.Seed preparation
The eds were prepared in the same manner as in the germi-nation experiment.The prepared eds of each test species were
J.Bae et al./Environmental Pollution213(2016)112e118113
placed in petri dishes containing6ml of0.8%(w/v)Bacto agar media(DIFCO).The dishes were aled with Parafilm to prevent evaporation and transferred to a growth chamber with the same condition as the germination experiment.The eds were allowed to germinate for48h.
2.4.2.Seedling growth substrate
All treatment solutions were solidified with0.8%(w/v)Bacto agar and adjusted to pH5.8prior to autoclaving.The treatment media(40ml)was transferred into a glass tube(50ml).Compared to potting media,agar medium growth substrate maintains target concentrations more readily,and it also ensures more rapid data collection and more reliable measurements(Di Salvatore et al., 2008).We found no effect of the itric acid,potas-sium hydroxide,nitrate,and potassium)on edlin
g growth and survival of the test species(unpublished data).
2.4.
3.Seedling transplant and growth condition
Uniform-sized edlings of each test species were transplanted into the tubes containing the treatment media(a single edling per tube).Five edlings of each test species for each metal con-centration were assigned in four plastic tube racks in a completely randomized design,and three repetitions were performed for each heavy metal.The tubes were transferred to a growth chamber with the same condition as the germination experiment.A transparent plastic cover(34Â16Â20cm)was placed over each tube rack to prevent the media from evaporating.The edlings were sprayed with deionized water every two days during the experiment period.
2.4.4.Seedling respon measurement
Three metal tolerance parameters were measured:IC50above and IC50below(the inhibitory concentration decreasing aboveground or belowground dry biomass by50%after three weeks);and LC50(the lethal concentration causing50%edling mortality within three weeks).Seedling mortality r
ate refers to percentage of edling death through visual inspection of total wilting,chlorosis,and ne-crosis at the end of the experiment.For biomass measurement, edlings were harvested after three weeks,gently rind with running distilled water,parated into above-and below-agar parts and dried at70 C to a constant weight.
2.5.Statistical analysis
Percentage final germination percentage)were arcsine transformed to improve normality and homogeneity of variance. One-way ANOVA was ud to investigate effects of the metals on germination rate andfinal germination percentage of the test species.The Tukey e Kramer HSD test was ud to compare treat-ment means where significant(P<0.05)differences were found with the ANOVA.
Each respon variables of the test dry biomass and mortality rate)and each metal concentration werefitted to linear [Y¼aXþb]and polynomial[Y¼a(XÀ50)2þbXþc for Zn,Pb,Ni, and Cu;Y¼a(XÀ5)2þbXþc)for Cd]models.Percentage edling mortality rate)were arcsine transformed beforefitting them to the model.Bad on R2and P values,the model(either linear or polynomial)bestfit to the data was ud to estimate each tolerance threshold IC50and LC50)
using inver pre-diction.All analys and estimations were conducted with JMP (Version10,SAS Institute Inc.,Cary,NC,USA).3.Results and discussion
3.1.Seed germination
In the cour of imbibition and germination,ed coats will eventually soften and be more permeable to various stress (Kranner and Colville,2011;Wierzbicka and Obidzi n ska,1998).The germination stage is thefirst exchange interface with the sur-rounding medium and accordingly it is relatively nsitive to changing environmental conditions(Shah et al.,2010;Solanki and Dhankhar,2011).The prent study showed that inhibitory effects of all the metals were greatest on T.arven germination(Table1). Compared to the control,TG of T.arven was decread by19and 61%at100and200mg Zn/kg,respectively.Under Pb and Cu at 50mg/kg,TG of T.arven was reduced by6and40%,but decread by!40and!70%at higher Pb and Cu levels.All Ni additions decread TG of T.arven by!70%,while Cd treatments caud about40%reduction in its TG.Additionally,T.arven germination was delayed by all metal additions.Compared to the control,T50of T.arven was delayed by Zn(!50%),Pb(!140%),Cu(!140%)and Cd(!150%),and the species did not reach50%of its TG when subjected to Zn at200mg/kg,Pb at200mg/kg,Ni at!50mg/kg and Cu at!100mg/kg(Table1).
The degree to which metal toxicity inhibits germination varies depending on the metal elements and the plant species and some species can tolerate heavy metal levels that are toxic to other nsitive species(Kranner and Colville,2011).We found that germination of A.artemisiifolia appeared to be more tolerant to the studied metals than T.arven.No effect of Zn,Pb,Ni or Cd on A.artemisiifolia germination was obrved(Table1).When sub-jected to Cu,TG of A.artemisiifolia was inhibited at a higher con-centration(!100mg Cu/kg)than T.arven(!50mg Cu/kg).The degree to which germination was delayed by!50mg Cu/kg was much lower in A.artemisiifolia(23%)than T.arven(143.5%)over the experimental period.Germination rate(T50)of A.artemisiifolia was promoted by24%at50mg Ni/kg and20%at50mg Pb/kg compared to the control(Table1).This is probably thefirst to report that low concentrations of Ni and Pb promote T50of A.artemisiifolia. Lef e vre et al.(2009)propod that a slightly enhanced level of oxidative ROS and RNS)could stimulate germination of metal tolerant species when expod to low metal concentrations. Bailly et al.(2008)reported that the ROS could play either beneficial (i.e.a positive signal for ed dormancy relea)or oxidative damage to biomolecules and cell death)role for ed germination.However,the exact physiological mechanism still remains unknown.No matter which mechanism is involved in the stimulation of germination rate,the greater ability of A.artemisiifolia to cope with some of the major metals in roadside soils can be one of its competitive advantages over current sup-plement covers such as T.arven.
Becau of their adaptive characteristics to veral abiotic drought,low fertility,and high salinity),C.varia iculatus are recommended as supplement ground cover along major roadways in many regions of North America(Escaray et al.,2012;Gover et al.,2007;NCDOT,1998).Thus,their germi-nation capacities under metal stress need to be examined to evaluate their potential for alternative supplement cover along roadsides of major roads in southern Qu e bec.There was no inhib-itory effect of Zn and Pb on TG of C.varia iculatus (Table1).Germination rate(T50)of C.varia was delayed by Zn and Pb additions,while iculatus was incread by1.7-fold at 200mg Pb/kg.At50,100and200mg Cu/kg,TG of C.varia was decread by13,41and82%,respectively,while TG iculatus was reduced by22,44and72%,respectively.There was no effect of Ni on TG and iculatus,but Ni at
J.Bae et al./Environmental Pollution213(2016)112e118 114
!100mg/kg inhibited TG and T50of C.varia(Table1).At5and 10mg Cd/kg,TG of C.varia was reduced by17and23%,respectively, but its T50was not affected by Cd(Table1).iculatus,Cd additions decread TG by26%,and delayed T50by!80%(Table1). Out results indicated that germination iculatus was tolerant to Zn,Pb and Ni additions.
3.2.Seedling growth
The edling stage is more vulnerable to metal stress than latter vegetative stages(Lef e vre et al.,2009;Shah et al.,2010).Testing early edling growth and mortality associated with metal stress can be another measure to evaluate a species establishment po-tential in metal-contaminated environments like roadside edges. Little variation in IC50above for Zn,Ni,and Cu among the test species indicated similar nsitivity to the metals in terms of aboveground biomass accumulation(Table2).No Pb addition caud!50%de-creas in aboveground biomass iculatus or T.arven edlings,whereas IC50above for A.artemisiifolia and C.varia were 54.2and67.2mg Pb/kg,respectively.It suggests that aboveground edling growths iculatus and T.arven were less nsitive to Pb than the other species.No Cd addition caud!50%decreas in aboveground biomass of A.artemisiifolia,while IC50above for other species iculatus(8.9mg Cd/kg),C.varia(3.7mg Cd/kg),and T.arven(5.4mg Cd/kg).Aboveground edling growth of A.artemisiifolia iculatus was more tolerant to Cd than the other species.Consistent with ourfindings,Abe et al. (2006)obrved that IC50above for Cd were19.7mg/kg for A.artemisiifolia and4.4mg/kg for T.arven.
The narrow range of IC50below implied that inhibitory effects of all metals on belowground growth were analogous among the test species(Table3).Given that edling roots are thefirst contact point w
ith toxic elements,reductions in root growth are usually the first visual effect of metal phytotoxicity and inhibitory effects of metals on root growth are more vere than shoot growth(Shah et al.,2010;Yang et al.,2010).In accordance with previousfind-ings,the lower values of IC50below than IC50above in the prent study indicated that belowground edling growth of the test species was more nsitive to the studied metals(Tables2and3). The different degrees of inhibition between the two parts probably resulted from different distribution of heavy metals in plants.Since heavy metals are found trapped in the cell walls and near to the uptake site,most of the metal(75e90%)taken up is largely found in the roots and smaller amounts are distributed in the shoots(Greger, 2004).
3.3.Seedling mortality
The low value of LC50reflected that the elevated Zn level (!40mg/kg)reduced edling survival of T.arven.In contrast,the higher LC50values(>100mg Zn/kg)of A.artemisiifolia,C.varia,iculatus indicated that Zn at high concentrations did not cau a lethal effect on their edlings.The effect of Pb additions on edling mortality of the test species was least among the studied metals(Table4),and this may result from the fact that Pb is the most immobile of the heavy metals from soil to shoot(Kabata-Pendias,2001;Kumar and Singh,1993).In contrast to Pb effects, more than80%edlings iculatus,C.varia,and T.arven died when subjected to the Ni and
Cu at!50mg/kg,and their nsitivities to the two metals were similar as indicated by the narrow range of LC50values(Table4).However,edling mortality rates of A.artemisiifolia under Ni and Cu at100mg/kg were27and 13%,respectively,which indicates its ability to tolerate to the metals than the other species during early growth pha.Seedlings of T.arven were obrved to be nsitive to Cd,while tho of A.artemisiifolia iculatus were tolerant as evidenced by
Table1
Effects of Zn,Pb,Ni,Cu,and Cd onfinal germination percentage(TG)and germination rate(T50)of the test plant species(Ambrosia artemisiifolia,Coronilla varia,Lotus cor-niculatus,and Trifolium arven)after two-week exposure.
HM Species Treatment(mg/kg)
050100200050100200
Final germination percentage(TG)(%)Germination rate(T50)(Hours)
Zn A.artemisiifolia90.6±2.0a a96.0±0.0a92.0±2.3a92.7±2.8a99.0±6.3a92.0±7.6a80.6±6.8a85.7±2.2a
C.varia80.0±3.7a76.0±3.7a78.7±5.6a70.0±3.1a74.4±4.2a113.3±6.1b93.5±6.1ab104.6±7.2b
Pb A.artemisiifolia86.7±1.3a84.0±3.7a88.7±1.6a83.3±1.2a132.0±5.8a106.0±8.6b109.0±3.8ab100.8±6.7b
C.varia78.0±3.1a74.7±1.3a80.0±1.5a72.0±2.9a80.0±7.2a110.0±5.2b102.4±5.4ab118.7±7.1b
异地恋情侣暖心小故事
Ni A.artemisiifolia82.7±2.9a84.7±2.4a77.3±2.5a84.7±3.8a121.0±4.9a90.8±5.1b108.0±6.4ab102.9±6.6ab
C.varia82.0±4.5a70.0±3.4ab61.3±2.0b64.0±2.9b62.2±4.3a95.6±9.8ab149.0±31.8b139.8±24.3b
Cu A.artemisiifolia90.7±2.0a86.0±1.4a66.0±0.9b44.7±1.2c96.7±7.2a119.2±1.9b119.2±1.9b>336
C.varia80.0±3.0a70.0±2.5b47.3±0.7c14.7±0.8d74.4±4.2a136.6±4.8b>336>336
池昌旭图片
T.arven96.0±1.5a56.7±1.9b18.7±1.3c11.3±1.9c41.8±1.4a101.8±7.1b>336>336
HM Species Treatment(mg/kg)
05100510
Final germination percentage(TG)(%)Germination rate(T50)(Hours)
Cd A.artemisiifolia93.3±2.0a a92.7±3.0a90.0±1.7a99.5±8.9a115.7±15.3a112.2±10.2a
C.varia82.0±2.5a68.0±3.3b63.3±4.1b73.7±5.2a115.7±14.6a139.7±32.2a酒店开业
T.arven95.3±2.6a63.3±2.8b54.7±1.3b43.0±1.1a97.6±4.2b112.8±5.2c
a Values(mean±SE)within rows not sharing the same letter are significantly different(Tukey e Kramer HSD;p<0.05).
b When thefinal germination percentage did not reach50%,T
50value(hours)was reprented as>336.
J.Bae et al./Environmental Pollution213(2016)112e118115
